Cyclic dinucleotides for treating conditions associated with sting activity such as cancer

ABSTRACT

This disclosure features dinucleotide compounds that modulate Stimulator of Interferon Genes (STING) activity, for use for example in the treatment of cancer. This disclosure also features compositions as well as other methods of using and making the same (Formula (A)). A and B are each independently selected from the group consisting of Formulae (i), (ii), (iii), and (iv).

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/277,273, filed on Jan. 11, 2016 and U.S. Provisional Application No.62/436,759, filed on Dec. 20, 2016, each of which is incorporated hereinby reference in its entirety.

TECHNICAL FIELD

This disclosure features chemical entities (e.g., a compound thatmodulates (e.g., agonizes) Stimulator of Interferon Genes (STING), or apharmaceutically acceptable salt, and/or hydrate, and/or cocrystal,and/or drug combination of the compound) that are useful, e.g., fortreating a condition, disease or disorder in which a decrease orincrease in STING activity (e.g., a decrease, e.g., a condition, diseaseor disorder associated with repressed or impaired STING signaling)contributes to the pathology and/or symptoms and/or progression of thecondition, disease or disorder (e.g., cancer) in a subject (e.g., ahuman). This disclosure also features compositions as well as othermethods of using and making the same.

BACKGROUND

STING, also known as transmembrane protein 173 (TMEM173) andMPYS/MITA/ERIS, is a protein that in humans is encoded by the TMEM173gene. STING has been shown to play a role in innate immunity. STINGinduces type I interferon production when cells are infected withintracellular pathogens, such as viruses, mycobacteria and intracellularparasites. Type I interferon, mediated by STING, protects infected cellsand nearby cells from local infection in an autocrine and paracrinemanner. The STING pathway is a pathway that is involved in the detectionof cytosolic DNA.

The STING signaling pathway is activated by cyclic dinucleotides (CDNs),which may be produced by bacteria or produced by antigen presentingcells in response to sensing cytosolic DNA. Unmodified CDNs have beenshown to induce type I interferon and other co-regulated genes, which inturn facilitate the development of a specific immune response (see,e.g., Wu and Sun, et al., Science 2013, 339, 826-830). WO 2015/077354discloses the use of STING agonists for the treatment of cancer.

SUMMARY

This disclosure features chemical entities (e.g., a compound thatmodulates (e.g., agonizes) Stimulator of Interferon Genes (STING), or apharmaceutically acceptable salt, and/or hydrate, and/or cocrystal,and/or drug combination of the compound) that are useful, e.g., fortreating a condition, disease or disorder in which a decrease orincrease in STING activity (e.g., a decrease, e.g., a condition, diseaseor disorder associated with repressed or impaired STING signaling)contributes to the pathology and/or symptoms and/or progression of thecondition, disease or disorder (e.g., cancer) in a subject (e.g., ahuman). In certain embodiments, the chemical entities described hereininduce an immune response in a subject (e.g., a human). In certainembodiments, the chemical entities described herein induceSTING-dependent type I interferon production in a subject (e.g., ahuman). This disclosure also features compositions as well as othermethods of using and making the same.

An “agonist” of STING includes compounds that, at the protein level,directly bind or modify STING such that an activity of STING isincreased, e.g., by activation, stabilization, altered distribution, orotherwise.

Certain compounds described herein that agonize STING to a lesser extentthan a STING full agonist can function in assays as antagonists as wellas agonists. These compounds antagonize activation of STING by a STINGfull agonist because they prevent the full effect of STING interaction.However, the compounds also, on their own, activate some STING activity,typically less than a corresponding amount of the STING full agonist.Such compounds may be referred to as “partial agonists of STING”.

In some embodiments, the compounds described herein are agonists (e.g.full agonists) of STING. In other embodiments, the compounds describedherein are partial agonists of STING.

Generally, a receptor exists in an active (Ra) and an inactive (Ri)conformation. Certain compounds that affect the receptor can alter theratio of Ra to Ri (Ra/Ri). For example, a full agonist increases theratio of Ra/Ri and can cause a “maximal”, saturating effect. A partialagonist, when bound to the receptor, gives a response that is lower thanthat elicited by a full agonist (e.g., an endogenous agonist). Thus, theRa/Ri for a partial agonist is less than for a full agonist. However,the potency of a partial agonist may be greater or less than that of thefull agonist.

While not wishing to be bound by theory, it is believed that the partialagonists of STING described herein provide advantages with regard totreating the disorders described herein. By way of example, the partialagonists of STING described herein exhibit intrinsic activities that areexpected to be both (i) high enough to induce an anti-tumor response(i.e., kill one or more tumor cells) and (ii) low enough to reduce thelikelihood of producing toxicity-related side effects. As discussedabove, partial agonists can antagonize activation of STING by a STINGfull agonist because they prevent the full effect of STING interaction,thereby reducing the activity of the STING full agonist. It is believedthat this antagonism can also modulate (e.g., reduce) the toxicityprofile of the STING full agonist. Accordingly, this disclosurecontemplates methods in which the partial agonists of STING describedherein are combined with one (or more) full agonists of STING (e.g., asdescribed anywhere herein) to provide therapeutic drug combinations thatare both efficacious and exhibit relatively low toxicity.

In one aspect, compounds of Formula A, or a pharmaceutically acceptablesalt thereof, are featured:

in which A, B, X, X′, G¹, G², X¹, X², X³, X⁴, X⁵, X⁶, L¹, L², R_(1A),R_(1B), R_(2A), and R_(2B) can be as defined anywhere herein. X¹ and X⁵can each be independently “up” or “down.”

In another aspect, compounds of Formula B, or a pharmaceuticallyacceptable salt thereof, are featured:

in which A, B, X, X′, G¹, G², X¹, X², X³, X⁴, X⁵, X⁶, L¹, L², R^(1A),R^(1B), R^(2A), and R_(2B) can be as defined anywhere herein. X¹ and X⁵can each be independently “up” or “down.”

In one aspect, compounds of Formula I, or a pharmaceutically acceptablesalt thereof, are featured:

in which A, B, X, X′, G¹, G², X¹, X², X³, X⁴, X⁵, X⁶, L¹, L², R_(1A),R_(1B), R_(2A), and R_(2B) can be as defined anywhere herein.

In one aspect, compounds of Formula A′, or a pharmaceutically acceptablesalt thereof, are featured:

in which A, B, X, X′, X¹, X², X³, X⁴, X⁵, X⁶, L¹, L², R_(1A), R_(1B),R_(2A), and R_(2B) can be as defined anywhere herein. X¹ and X⁵ can eachbe independently “up” or “down.”

In another aspect, compounds of Formula B′, or a pharmaceuticallyacceptable salt thereof, are featured:

in which A, B, X, X′, X¹, X², X³, X⁴, X⁵, X⁶, L¹, L², R_(1A), R_(1B),R_(2A), and R_(2B) can be as defined anywhere herein. X¹ and X⁵ can eachbe independently “up” or “down.”

In another aspect, compounds of Formula I-A, or a pharmaceuticallyacceptable salt thereof, are featured:

in which A, B, X, X′, X¹, X², X³, X⁴, X⁵, X⁶, L¹, L², R_(1A), R_(1B),R_(2A), and R_(2B) can be as defined anywhere herein.

In one aspect, pharmaceutical compositions are featured that include achemical entity described herein (e.g., a compound described genericallyor specifically herein or a pharmaceutically acceptable salt thereof orcompositions containing the same) and one or more pharmaceuticallyacceptable excipients.

In one aspect, methods for modulating (e.g., agonizing) STING activityare featured that include contacting STING with a chemical entitydescribed herein (e.g., a compound described generically or specificallyherein or a pharmaceutically acceptable salt thereof or compositionscontaining the same). Methods include in vitro methods, e.g., contactinga sample that includes one or more cells comprising STING (e.g., innateimmune cells, e.g., mast cells, macrophages, dendritic cells (DCs), andnatural killer cells) with the chemical entity. The contacting can, insome cases, induce an immune response sufficient to kill at least one ofthe one or more cancer cells. Methods can also include in vivo methods;e.g., administering the chemical entity to a subject (e.g., a human)having a disease in which repressed or impaired STING signalingcontributes to the pathology and/or symptoms and/or progression of thedisease (e.g., cancer; e.g., a refractory cancer).

In another aspect, methods of treating cancer are featured that includeadministering to a subject in need of such treatment an effective amountof a chemical entity described herein (e.g., a compound describedgenerically or specifically herein or a pharmaceutically acceptable saltthereof or compositions containing the same).

In a further aspect, methods of inducing an immune response (e.g., aninnate immune response) in a subject in need thereof are featured thatinclude administering to the subject an effective amount of a chemicalentity described herein (e.g., a compound described generically orspecifically herein or a pharmaceutically acceptable salt thereof orcompositions containing the same).

In another aspect, methods of inducing induce STING-dependent type Iinterferon production in a subject in need thereof are featured thatinclude administering to the subject an effective amount of a chemicalentity described herein (e.g., a compound described generically orspecifically herein or a pharmaceutically acceptable salt thereof orcompositions containing the same).

In a further aspect, methods of treatment of a disease in whichrepressed or impaired STING signaling contributes to the pathologyand/or symptoms and/or progression of the disease are featured thatinclude administering to a subject in need of such treatment aneffective amount of a chemical entity described herein (e.g., a compounddescribed generically or specifically herein or a pharmaceuticallyacceptable salt thereof or compositions containing the same).

In another aspect, methods of treatment are featured that includeadministering to a subject having a disease in which repressed orimpaired STING signaling contributes to the pathology and/or symptomsand/or progression of the disease an effective amount of a chemicalentity described herein (e.g., a compound described generically orspecifically herein or a pharmaceutically acceptable salt thereof orcompositions containing the same).

In a further aspect, methods of treatment that include administering toa subject a chemical entity described herein (e.g., a compound describedgenerically or specifically herein or a pharmaceutically acceptable saltthereof or compositions containing the same), wherein the chemicalentity is administered in an amount effective to treat a disease inwhich repressed or impaired STING signaling contributes to the pathologyand/or symptoms and/or progression of the disease, thereby treating thedisease.

Embodiments can include one or more of the following features.

The chemical entity can be administered in combination with one or moreadditional cancer therapies (e.g., surgery, radiotherapy, chemotherapy,toxin therapy, immunotherapy, cryotherapy or gene therapy, or acombination thereof; e.g., chemotherapy that includes administering oneor more (e.g., two, three, four, five, six, or more) additionalchemotherapeutic agents. Non-limiting examples of additionalchemotherapeutic agents is selected from an alkylating agent (e.g.,cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil,ifosfamide and/or oxaliplatin); an anti-metabolite (e.g., azathioprineand/or mercaptopurine); a terpenoid (e.g., a vinca alkaloid and/or ataxane; e.g., Vincristine, Vinblastine, Vinorelbine and/or VindesineTaxol, Pacllitaxel and/or Docetaxel); a topoisomerase (e.g., a type Itopoisomerase and/or a type 2 topoisomerase; e.g., camptothecins, suchas irinotecan and/or topotecan; amsacrine, etoposide, etoposidephosphate and/or teniposide); a cytotoxic antibiotic (e.g., actinomycin,anthracyclines, doxorubicin, daunorubicin, valrubicin, idarubicin,epirubicin, bleomycin, plicamycin and/or mitomycin); a hormone (e.g., alutenizing hormone releasing hormone agonist; e.g., leuprolidine,goserelin, triptorelin, histrelin, bicalutamide, flutamide and/ornilutamide); an antibody (e.g., Abciximab, Adalimumab, Alemtuzumab,Atlizumab, Basiliximab, Belimumab, Bevacizumab, Bretuximab vedotin,Canakinumab, Cetuximab, Ceertolizumab pegol, Daclizumab, Denosumab,Eculizumab, Efalizumab, Gemtuzumab, Golimumab, Golimumab, Ibritumomabtiuxetan, Infliximab, Ipilimumab, Muromonab-CD3, Natalizumab,Ofatumumab, Omalizumab, Palivizumab, Panitumuab, Ranibizumab, Rituximab,Tocilizumab, Tositumomab and/or Trastuzumab); an anti-angiogenic agent;a cytokine; a thrombotic agent; a growth inhibitory agent; ananti-helminthic agent; and an immune checkpoint inhibitor that targetsan immune checkpoint receptor selected from the group consisting ofCTLA-4, PD-1, PD-L1, PD-1-PD-L1, PD-1-PD-L2, interleukin-2 (IL-2),indoleamine 2,3-dioxygenase (IDO), IL-10, transforming growth factor-β(TGFβ), T cell immunoglobulin and mucin 3 (TIM3 or HAVCR2), Galectin9-TIM3, Phosphatidylserine-TIM3, lymphocyte activation gene 3 protein(LAG3), MHC class II-LAG3, 4-1BB-4-1BB ligand, OX40-OX40 ligand, GITR,GITR ligand-GITR, CD27, CD70-CD27, TNFRSF25, TNFRSF25-TL1A, CD40L,CD40-CD40 ligand, HVEM-LIGHT-LTA, HVEM, HVEM-BTLA, HVEM-CD160,HVEM-LIGHT, HVEM-BTLA-CD160, CD80, CD80-PDL-1, PDL2-CD80, CD244,CD48-CD244, CD244, ICOS, ICOS-ICOS ligand, B7-H3, B7-H4, VISTA, TMIGD2,HHLA2-TMIGD2, Butyrophilins, including BTNL2, Siglec family, TIGIT andPVR family members, KIRs, ILTs and LIRs, NKG2D and NKG2A, MICA and MICB,CD244, CD28, CD86-CD28, CD86-CTLA, CD80-CD28, CD39, CD73Adenosine-CD39-CD73, CXCR4-CXCL12, Phosphatidylserine, TIM3,Phosphatidylserine-TIM3, SIRPA-CD47, VEGF, Neuropilin, CD160, CD30, andCD155 (e.g., CTLA-4 or PD1 or PD-L1).

The subject can have cancer; e.g., the subject has undergone and/or isundergoing and/or will undergo one or more cancer therapies.

Non-limiting examples of cancer include melanoma, cervical cancer,breast cancer, ovarian cancer, prostate cancer, testicular cancer,urothelial carcinoma, bladder cancer, non-small cell lung cancer, smallcell lung cancer, sarcoma, colorectal adenocarcinoma, gastrointestinalstromal tumors, gastroesophageal carcinoma, colorectal cancer,pancreatic cancer, kidney cancer, hepatocellular cancer, malignantmesothelioma, leukemia, lymphoma, myelodysplasia syndrome, multiplemyeloma, transitional cell carcinoma, neuroblastoma, plasma cellneoplasms, Wilm's tumor, or hepatocellular carcinoma. In certainembodiments, the cancer can be a refractory cancer.

The chemical entity can be administered intratumorally.

The methods can further include identifying the subject.

Other embodiments include those described in the Detailed Descriptionand/or in the claims.

Additional Definitions

To facilitate understanding of the disclosure set forth herein, a numberof additional terms are defined below. Generally, the nomenclature usedherein and the laboratory procedures in organic chemistry, medicinalchemistry, and pharmacology described herein are those well-known andcommonly employed in the art. Unless defined otherwise, all technicaland scientific terms used herein generally have the same meaning ascommonly understood by one of ordinary skill in the art to which thisdisclosure belongs. Each of the patents, applications, publishedapplications, and other publications that are mentioned throughout thespecification and the attached appendices are incorporated herein byreference in their entireties.

As used herein, the term “STING” is meant to include, withoutlimitation, nucleic acids, polynucleotides, oligonucleotides, sense andantisense polynucleotide strands, complementary sequences, peptides,polypeptides, proteins, homologous and/or orthologous STING molecules,isoforms, precursors, mutants, variants, derivatives, splice variants,alleles, different species, and active fragments thereof.

The term “acceptable” with respect to a formulation, composition oringredient, as used herein, means having no persistent detrimentaleffect on the general health of the subject being treated.

“API” refers to an active pharmaceutical ingredient.

The terms “effective amount” or “therapeutically effective amount,” asused herein, refer to a sufficient amount of a chemical entity (e.g., acompound exhibiting activity as a mitochondrial uncoupling agent or apharmaceutically acceptable salt and/or hydrate and/or cocrystalthereof; e.g., a compound, such as niclosamide or a pharmaceuticallyacceptable salt and/or hydrate and/or cocrystal thereof; e.g., acompound, such as a niclosamide analog, or a pharmaceutically acceptablesalt and/or hydrate and/or cocrystal thereof) being administered whichwill relieve to some extent one or more of the symptoms of the diseaseor condition being treated. The result includes reduction and/oralleviation of the signs, symptoms, or causes of a disease, or any otherdesired alteration of a biological system. For example, an “effectiveamount” for therapeutic uses is the amount of the composition comprisinga compound as disclosed herein required to provide a clinicallysignificant decrease in disease symptoms. An appropriate “effective”amount in any individual case is determined using any suitabletechnique, such as a dose escalation study.

The term “excipient” or “pharmaceutically acceptable excipient” means apharmaceutically-acceptable material, composition, or vehicle, such as aliquid or solid filler, diluent, carrier, solvent, or encapsulatingmaterial. In one embodiment, each component is “pharmaceuticallyacceptable” in the sense of being compatible with the other ingredientsof a pharmaceutical formulation, and suitable for use in contact withthe tissue or organ of humans and animals without excessive toxicity,irritation, allergic response, immunogenicity, or other problems orcomplications, commensurate with a reasonable benefit/risk ratio. See,e.g., Remington: The Science and Practice of Pharmacy, 21st ed.;Lippincott Williams & Wilkins: Philadelphia, Pa., 2005; Handbook ofPharmaceutical Excipients, 6th ed.; Rowe et al., Eds.; ThePharmaceutical Press and the American Pharmaceutical Association: 2009;Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; GowerPublishing Company: 2007; Pharmaceutical Preformulation and Formulation,2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, Fla., 2009.

The term “pharmaceutically acceptable salt” refers to a formulation of acompound that does not cause significant irritation to an organism towhich it is administered and does not abrogate the biological activityand properties of the compound. In certain instances, pharmaceuticallyacceptable salts are obtained by reacting a compound described herein,with acids such as hydrochloric acid, hydrobromic acid, sulfuric acid,nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid,p-toluenesulfonic acid, salicylic acid and the like. In some instances,pharmaceutically acceptable salts are obtained by reacting a compoundhaving acidic group described herein with a base to form a salt such asan ammonium salt, an alkali metal salt, such as a sodium or a potassiumsalt, an alkaline earth metal salt, such as a calcium or a magnesiumsalt, a salt of organic bases such as dicyclohexylamine,N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts withamino acids such as arginine, lysine, and the like, or by other methodspreviously determined. The pharmacologically acceptable salt s notspecifically limited as far as it can be used in medicaments. Examplesof a salt that the compounds described hereinform with a base includethe following: salts thereof with inorganic bases such as sodium,potassium, magnesium, calcium, and aluminum; salts thereof with organicbases such as methylamine, ethylamine and ethanolamine; salts thereofwith basic amino acids such as lysine and ornithine; and ammonium salt.The salts may be acid addition salts, which are specifically exemplifiedby acid addition salts with the following: mineral acids such ashydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid,nitric acid, and phosphoric acid:organic acids such as formic acid,acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid,fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid,citric acid, methanesulfonic acid, and ethanesulfonic acid; acidic aminoacids such as aspartic acid and glutamic acid.

The term “pharmaceutical composition” refers to a mixture of a compounddescribed herein with other chemical components (referred tocollectively herein as “excipients”), such as carriers, stabilizers,diluents, dispersing agents, suspending agents, and/or thickeningagents. The pharmaceutical composition facilitates administration of thecompound to an organism. Multiple techniques of administering a compoundexist in the art including, but not limited to: rectal, oral,intravenous, aerosol, parenteral, ophthalmic, pulmonary, and topicaladministration.

The term “subject” refers to an animal, including, but not limited to, aprimate (e.g., human), monkey, cow, pig, sheep, goat, horse, dog, cat,rabbit, rat, or mouse. The terms “subject” and “patient” are usedinterchangeably herein in reference, for example, to a mammaliansubject, such as a human.

The terms “treat,” “treating,” and “treatment,” in the context oftreating a disease or disorder, are meant to include alleviating orabrogating a disorder, disease, or condition, or one or more of thesymptoms associated with the disorder, disease, or condition; or toslowing the progression, spread or worsening of a disease, disorder orcondition or of one or more symptoms thereof. The “treatment of cancer”,refers to one or more of the following effects: (1) inhibition, to someextent, of tumor growth, including, (i) slowing down and (ii) completegrowth arrest; (2) reduction in the number of tumor cells; (3)maintaining tumor size; (4) reduction in tumor size; (5) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of tumor cell infiltration into peripheral organs; (6) inhibition,including (i) reduction, (ii) slowing down or (iii) complete prevention,of metastasis; (7) enhancement of anti-tumor immune response, which mayresult in (i) maintaining tumor size, (ii) reducing tumor size, (iii)slowing the growth of a tumor, (iv) reducing, slowing or preventinginvasion and/or (8) relief, to some extent, of the severity or number ofone or more symptoms associated with the disorder.

The term “halo” refers to fluoro (F), chloro (Cl), bromo (Br), or iodo(I).

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁₋₁₀ indicates that the group may have from 1 to 10(inclusive) carbon atoms in it. Non-limiting examples include methyl,ethyl, iso-propyl, tert-butyl, n-hexyl.

The term “haloalkyl” refers to an alkyl, in which one or more hydrogenatoms is/are replaced with an independently selected halo.

The term “alkoxy” refers to an —O-alkyl radical (e.g., —OCH₃).

The term “alkylene” refers to a divalent alkyl (e.g., —CH₂—).

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂₋₆ indicates that the group may have from 2 to 6 (inclusive)carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂₋₆ indicates that the group may have from 2 to 6 (inclusive)carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic, 10-carbon bicyclic, or14-carbon tricyclic aromatic ring system wherein 0, 1, 2, 3, or 4 atomsof each ring may be substituted by a substituent. Examples of arylgroups include phenyl, naphthyl and the like.

The term “cycloalkyl” as used herein includes saturated cyclichydrocarbon groups having 3 to 10 carbons, preferably 3 to 8 carbons,and more preferably 3 to 6 carbons, wherein the cycloalkyl group may beoptionally substituted. Preferred cycloalkyl groups include, withoutlimitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl,cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

In addition, atoms making up the compounds of the present embodimentsare intended to include all isotopic forms of such atoms. Isotopes, asused herein, include those atoms having the same atomic number butdifferent mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include ¹³C and ¹⁴C.

The details of one or more embodiments of the invention are set forth inthe description below. Other features and advantages of the inventionwill be apparent from the description, and from the claims.

DETAILED DESCRIPTION

This disclosure features chemical entities (e.g., a compound thatmodulates (e.g., agonizes) Stimulator of Interferon Genes (STING), or apharmaceutically acceptable salt, and/or hydrate, and/or cocrystal,and/or drug combination of the compound) that are useful, e.g., fortreating a condition, disease or disorder in which a decrease orincrease in STING activity (e.g., a decrease, e.g., a condition, diseaseor disorder associated with repressed or impaired STING signaling)contributes to the pathology and/or symptoms and/or progression of thecondition, disease or disorder (e.g., cancer) in a subject (e.g., ahuman). In certain embodiments, the chemical entities described hereininduce an immune response in a subject (e.g., a human). In certainembodiments, the chemical entities described herein induceSTING-dependent type I interferon production in a subject (e.g., ahuman). This disclosure also features compositions as well as othermethods of using and making the same.

Formula I Compounds

In one aspect, compounds of Formula A, or a pharmaceutically acceptablesalt thereof, are featured:

wherein:

A and B are each independently selected from the group consisting ofFormulae (i), (ii), (iii), and (iv):

X and X′ are each independently selected from the group consisting of O,S, S(O), SO₂, CH₂, CHF, CF₂, CH₂O, OCH₂, CH₂CH₂, CH═CH, NR³, andN(O⁻)R³;

G¹ is a bond connecting (i) the carbon directly attached to X² and (ii)the carbon directly attached to C(R^(2A))(R^(2B))(X⁶); or isC(R^(G1A))(R^(G1B));

G² is a bond connecting (i) the carbon directly attached to X⁴ and (ii)the carbon directly attached to C(R^(1A))(R^(1B))(X³); or isC(R^(G2A))(R^(G2B));

X¹ and X⁵ are each independently selected from the group consisting ofH, C₁₋₄ alkyl, C₁₋₄ haloalkyl, halo (e.g., F), —CN, —NO₂, —N₃, —OH,—OR^(a1), —SH, —SR^(a1), —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1),—C(O)OH, —C(O)OR^(a1), —OC(O)H, —OC(O)R^(a1), —OC(O)NR^(b1)R^(c1),—C(═NR^(e1))NR^(b1)R^(c1), —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1),—NR^(b1)R^(c1), —⁺NR^(b1)R^(c1)R^(d1), —NR^(d1)C(O)H,—NR^(d1)C(O)R^(a1), —NR^(d1)C(O)OR^(a1), —NR^(d1)C(O)NR^(b1)R^(c1),—NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), —NR^(d1)S(O)₂NR^(b1)R^(c1),—S(O)R^(a1), —S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1);

X², X³, X⁴ and X⁶ are each independently selected from the groupconsisting of O, S, and N—R³;

L¹ is

L² is

Y¹ and Y² are each independently selected from the group consisting of—OH, —OR^(a1), O⁻, —SH, —SR^(a1), S⁻; and —NR^(b1)R^(c1);

R^(1A) and R^(1B) are each independently selected from the groupconsisting of H; halo; C₁₋₄ alkyl; C₁₋₄ haloalkyl; C₂₋₄ alkenyl; C₂₋₄alkynyl; and C₃₋₅ cycloalkyl, which is optionally substituted with from1-4 independently selected C₁₋₄ alkyl; or R^(1A) and R^(1B), togetherwith the carbon atom to which each is attached, form a C₃₋₅ cycloalkylor heterocyclyl, including from 4-5 ring atoms, wherein from 1-2(e.g., 1) ring atoms are independently selected from the groupconsisting of nitrogen and oxygen (e.g., oxetane), wherein the C₃₋₅cycloalkyl or heterocyclyl ring can each be optionally substituted withfrom 1-4 independently selected C₁₋₄ alkyl;

R^(2A) and R^(2B) are each independently selected from the groupconsisting of H; halo; C₁₋₄ alkyl; C₁₋₄ haloalkyl; C₂₋₄ alkenyl; C₂₋₄alkynyl; and C₃₋₅ cycloalkyl, which is optionally substituted with from1-4 independently selected C₁₋₄ alkyl; or R^(2A) and R^(2B), togetherwith the carbon atom to which each is attached, form a C₃₋₅ cycloalkylor heterocyclyl, including from 4-5 ring atoms, wherein from 1-2(e.g., 1) ring atoms are independently selected from the groupconsisting of nitrogen and oxygen (e.g., oxetane), wherein the C₃₋₅cycloalkyl or heterocyclyl ring can each be optionally substituted withfrom 1-4 independently selected C₁₋₄ alkyl,

Z₁ is N or C—R⁴;

Z_(1′) is N or C—H;

Z₂ is N or C—R^(4′);

Z_(2′) is N or C—H;

Z₃ is N—R³ or C—R⁴;

each occurrence of R^(a1) is independently selected from the groupconsisting of:

-   -   C₁₋₁₀ alkyl optionally substituted with from 1-3 R^(A);    -   C₁₋₁₀ haloalkyl optionally substituted with from 1-3 R^(A);    -   C₂₋₁₀ alkenyl optionally substituted with from 1-3 R^(B),    -   C₂₋₁₀ alkynyl optionally substituted with from 1-3 R^(B),    -   C₃₋₁₀ cycloalkyl optionally substituted with from 1-5 R^(C);    -   (C₃₋₁₀ cycloalkyl)-C₁₋₆ alkylene, wherein the alkylene serves as        the point of attachment, and wherein the C₃₋₁₀ cycloalkyl        optionally substituted with from 1-5 R^(C);    -   heterocyclyl, including from 3-10 ring atoms, wherein from 1-3        ring atoms are independently selected from the group consisting        of nitrogen, oxygen and sulfur, and which is optionally        substituted with from 1-5 R^(C);    -   (heterocyclyl as defined above)-C₁₋₆ alkylene, wherein the        alkylene serves as the point of attachment, and wherein the        heterocyclyl is optionally substituted with from 1-5 R^(C);    -   C₆₋₁₀ aryl optionally substituted with from 1-5 R^(D);    -   heteroaryl including from 5-10 ring atoms, wherein from 1-4 ring        atoms are independently selected from the group consisting of        nitrogen, oxygen and sulfur, and which is optionally substituted        with from 1-5 R^(D); and    -   (heteroaryl as defined above)-C₁₋₆ alkylene, wherein the        alkylene serves as the point of attachment, and wherein the        heteroaryl optionally substituted with from 1-5 R^(D);

each occurrence of R³, R^(b1), R^(c1), R^(d1), and R^(e1) isindependently selected from the group consisting of: H; R^(a1); —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OR^(a1), —OC(O)H,—C(═NR^(e1))NR^(b1)R^(c1), —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1),—NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1), S(O)₂R^(a1), and—S(O)₂NR^(b1)R^(c1); or

R^(b1) and R^(c1) taken together with the nitrogen atom to which each isattached form a heterocyclyl, including from 3-10 ring atoms, whereinfrom 1-3 ring atoms are independently selected from the group consistingof nitrogen, oxygen and sulfur, and which is optionally substituted withfrom 1-5 R^(C); (e.g., azetidinyl, morpholino, piperidinyl);

each occurrence of R^(G1A), R^(G1B), R^(G1A) (R^(G2A)), R^(G1B)(R^(G2B)), R⁴, R^(4′), R⁵, R⁶, and R^(6′) is independently selected fromthe group consisting of: H; R^(a1); halo, —CN, —NO₂, —N₃, —OH, —OR^(a1),—SH, —SR^(a1), —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH,—C(O)OR^(a1), —OC(O)H, —OC(O)R^(a1), —OC(O)NR^(b1)R^(c1),—C(═NR^(e1))NR^(b1)R^(c1), —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1),—NR^(b1)R^(c1), —N⁺R^(b1)R^(c1)R^(d1), —NR^(d1)C(O)H,—NR^(d1)C(O)R^(a1), —NR^(c1)C(O)OR^(a1), —NR^(d1)C(O)NR^(b1)R^(c1),—NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), —NR^(d1)S(O)₂NR^(b1)R^(c1),—S(O)R^(a1), —S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1);

each occurrence of R^(A) is independently selected from the groupconsisting of: —CN; —OH; C₁₋₆ alkoxy; C₁₋₆ haloalkoxy; —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;—C(O)OH; —C(O)O(C₁₋₆ alkyl); and —NR″R′″, wherein R″ and R′″ are eachindependently selected from the group consisting of H, C₁₋₄ alkyl,—SO₂(C₁₋₆ alkyl), —C(O)(C₁₋₆ alkyl), and —C(O)O(C₁₋₆ alkyl);

each occurrence of R^(B) is independently selected from the groupconsisting of: halo; —CN; —OH; C₁₋₆ alkoxy; C₁₋₆ haloalkoxy; —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;—C(O)OH; —C(O)O(C₁₋₆ alkyl); and —NR″R″′, wherein R″ and R″′ are eachindependently selected from the group consisting of H, C₁₋₄ alkyl,—SO₂(C₁₋₆ alkyl), —C(O)(C₁₋₆ alkyl), and —C(O)O(C₁₋₆ alkyl);

each occurrence of R^(C) is independently selected from the groupconsisting of: C₁₋₆ alkyl; C₁₋₄ haloalkyl; halo; —CN; —OH; oxo; C₁₋₆alkoxy; C₁₋₆ haloalkoxy; —C(O)NRR′, wherein R′ and R″ are eachindependently selected from H and C₁₋₄ alkyl; —C(O)(C₁₋₆ alkyl);—C(O)OH; —C(O)O(C₁₋₆ alkyl); and —NR″R″′, wherein R″ and R″′ are eachindependently selected from the group consisting of H, C₁₋₄ alkyl,—SO₂(C₁₋₆ alkyl), —C(O)(C₁₋₆ alkyl), and —C(O)O(C₁₋₆ alkyl);

each occurrence of R^(D) is independently selected from the groupconsisting of:

-   -   C₁₋₆ alkyl optionally substituted with from 1-2 substituents        independently selected from the group consisting of: —OH, C₁₋₄        alkoxy; C₁₋₄ haloalkoxy; —NH₂, —NH(C₁₋₄ alkyl), and —N(C₁₋₄        alkyl)₂;    -   C₁₋₄ haloalkyl;    -   C₂₋₄ alkenyl;    -   C₂₋₄ alkynyl;    -   halo;    -   —CN;    -   —NO₂;    -   —N₃;    -   —OH;    -   C₁₋₆ alkoxy;    -   C₁₋₆ haloalkoxy;    -   —C(O)NRR′, wherein R′ and R″ are each independently selected        from H and C₁₋₄ alkyl;    -   —SO₂NRR′, wherein R′ and R″ are each independently selected from        H and C₁₋₄ alkyl;    -   —C(O)(C₁₋₆ alkyl);    -   —C(O)OH;    -   —C(O)O(C₁₋₆ alkyl);    -   —SO₂(C₁₋₆ alkyl),    -   —NR″R″′, wherein R″ and R″′ are each independently selected from        the group consisting of H, C₁₋₄ alkyl, —SO₂(C₁₋₆ alkyl),        —C(O)(C₁₋₆ alkyl), and —C(O)O(C₁₋₆ alkyl);    -   (C₃₋₁₀ cycloalkyl)-(CH₂)₀₋₂, wherein the CH₂ (when present)        serves as the point of attachment, and wherein the C₃₋₁₀        cycloalkyl is optionally substituted with from 1-5 independently        selected C₁₋₄ alkyl;    -   (heterocyclyl as defined above)-(CH₂)₀₋₂, wherein the CH₂ (when        present) serves as the point of attachment, and wherein the        heterocyclyl is optionally substituted with from 1-5        independently selected C₁₋₄ alkyl;    -   (phenyl)-(CH₂)₀₋₂, wherein the CH₂ (when present) serves as the        point of attachment, and wherein the phenyl is optionally        substituted with from 1-5 substituents independently selected        from halo, C₁₋₄ alkyl, —CF₃, —OCH₃, —SCH₃, —OCF₃, —NO₂, —N₃,        —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂, —C(O)(C₁₋₄ alkyl),        —C(O)OH, —C(O)O(C₁₋₄ alkyl), —SO₂(CH₃), and cyclopropyl; and    -   (heteroaryl as defined above)-(CH₂)₀₋₂, wherein the CH₂ (when        present) serves as the point of attachment, and wherein the        phenyl is optionally substituted with from 1-5 substituents        independently selected from halo, C₁₋₄ alkyl, —CF₃, —OCH₃,        —SCH₃, —OCF₃, —NO₂, —N₃, —NH₂, —NH(C₁₋₄ alkyl), —N(C₁₋₄ alkyl)₂,        —C(O)(C₁₋₄ alkyl), —C(O)OH, —C(O)O(C₁₋₄ alkyl), —SO₂(CH₃), and        cyclopropyl; and        provided at least one of X², X³, X⁴ and X⁶ is N—R³.        In some embodiments, it is further provided that the compound is        not:

Variables X, X′, G¹, and G²

In some embodiments, the compounds have formula (B). In someembodiments, the compounds have formula (I).

In some embodiments, X and X′ are each O. In some embodiments, G¹ is abond connecting (i) the carbon directly attached to X² and (ii) thecarbon directly attached to C(R^(2A))(R^(2B))(X⁶). In some embodiments,G² is a bond connecting (i) the carbon directly attached to X⁴ and (ii)the carbon directly attached to C(R^(1A))(R^(1B))(X³).

In some embodiments, X and X′ are each O, G¹ is a bond connecting (i)the carbon directly attached to X² and (ii) the carbon directly attachedto C(R^(2A))(R^(2B))(X⁶), G² is a bond connecting (i) the carbondirectly attached to X⁴ and (ii) the carbon directly attached toC(R^(1A))(R^(1B))(X³), and the compound has formula (A′), (B′), or (I-A)described previously.

Variables A and B

In some embodiments, A and B are each independently selected from thegroup consisting of formula (i) and formula (ii). In certainembodiments, A has formula (i), and B has formula (ii). In otherembodiments, A has formula (ii), and B has formula (ii). In still otherembodiments, A has formula (i), and B has formula (i).

In some embodiments, each occurrence of Z¹ is N, and Z^(1′) is N. Insome embodiments, R⁵ is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)). Insome embodiments, each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is—NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)). In certain of theseembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H.

In some embodiments, each occurrence of Z¹ is N, and Z^(1′) is N. Insome embodiments, R⁵ is —OH. In some embodiments, each occurrence of Z¹is N, Z^(1′) is N, and R⁵ is —OH. In certain of these embodiments, R⁶ isH. In certain of these embodiments, R⁴ is H; in other embodiments, R⁴ isother than H. For example, each occurrence of Z¹ is N; Z^(1′) is N; R⁵is —OH; R⁶ is H; and R⁴ is H.

In some embodiments, each occurrence of Z² is N, Z^(2′) is. N, and Z³ isN—R³ (e.g., N—H). In some embodiments, R^(6′) is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)). In some embodiments, each occurrence of Z² is N,Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), and R^(6′) is —NR^(b1)R^(c1)(e.g., —NH₂ or —NHR^(c1)). In certain of these embodiments, R^(4′) is H;in other embodiments, R^(4′) is other than H.

In certain of the foregoing embodiments, each occurrence of R^(b1) andR^(c1) or each occurrence of R^(c1) is independently selected from thegroup consisting of: H; R^(a1); —C(O)H, —C(O)R^(a1), —C(O)NRR′, whereinR and R′ are each independently selected from H and C₁₋₄ alkyl;—C(O)OR^(a1), —OC(O)H, —S(O)R^(a1), and —S(O)₂R^(a1).

In certain of the foregoing embodiments, each occurrence of R^(b1) andR^(c1) or each occurrence of R^(c1) is independently selected from thegroup consisting of: H; C₁₋₆ (e.g., C₁₋₄) alkyl optionally substitutedwith from 1-3 R^(A); —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyloptionally substituted with from 1-3 R^(A)); —C(O)NRR′, wherein R and R′are each independently selected from H and C₁₋₄ alkyl optionallysubstituted with from 1-3 R^(A); and —C(O)O(C₁₋₆ alkyl optionallysubstituted with from 1-3 R^(A)).

In certain of the foregoing embodiments, each occurrence of R^(b1) andR^(c1) or each occurrence of R^(c1) is independently selected from thegroup consisting of: H; C₁₋₆ (e.g., C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl);—C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′, wherein R and R′ are eachindependently selected from H and C₁₋₄ alkyl; and —C(O)O(C₁₋₆ alkyl).

Variables X², X³, X⁴ and X⁶

In some embodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³and X⁶) are N—R³ (e.g., N—H). In certain embodiments, X² and X⁴ are N—R³(e.g., N—H). In other embodiments, X³ and X⁶ are N—R³ (e.g., N—H).

In certain embodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³and X⁶) are N—R³ (e.g., N—H), and the others are O. In certainembodiments, X² and X⁴ are N—R³ (e.g., N—H), and the others are O. Inother embodiments, X³ and X⁶ are N—R³ (e.g., N—H), and the others are O.

Variables X¹ and X⁵

In some embodiments, X¹ is —OH, —OR^(a1), —F, —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), —NO₂, —N₃,—NR^(d1)C(═NR^(e1))NR^(b1)R^(c1), —NR^(b1)R^(c1), —⁺NR^(b1)R^(c1)R^(d1),—NR^(d1)C(O)H, —NR^(d1)C(O)R^(a1), —NR^(d1)C(O)OR^(a1),—NR^(d1)C(O)NR^(b1)R^(c1), —NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), or—NR^(d1)S(O)₂NR^(b1)R^(c1) (in certain embodiments, X¹ is other than—F).

In certain embodiments, X¹ is —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1),—OC(O)NR^(b1)R^(c1), —F, —NO₂, —N₃, —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1),—NR^(b1)R^(c1), —⁺NR^(b1)R^(c1)R^(d1), —NR^(d1)C(O)H,—NR^(d1)C(O)R^(a1), —NR^(d1)C(O)OR^(a1), —NR^(d1)C(O)NR^(b1)R^(c1),—NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), or —NR^(d1)S(O)₂NR^(b1)R^(c1)(in certain embodiments, X¹ is other than —F).

In certain embodiments, X¹ is —F, —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1),or —OC(O)NR^(b1)R^(c1) (in certain embodiments, X¹ is other than —F).

In certain embodiments, X¹ is —F, —OH or —OR^(a1) (in certainembodiments, X¹ is other than —F).

In certain embodiments, X¹ is —OH.

In certain embodiments, X¹ is —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1) (e.g., —OH or —OR^(a1); e.g., —OH), and two of X²,X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³ and X⁶) are N—R³ (e.g., N—H). Incertain embodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³and X⁶) are N—R³ (e.g., N—H), and the others are O.

In other embodiments, X¹ is —NO₂, —N₃, —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1),—NR^(b1)R—NR^(b1)R^(c1), —⁺NR^(b1)R^(c1)R^(d1), —NR^(d1)C(O)H,—NR^(d1)C(O)R^(a1), —NR^(d1)C(O)OR^(a1), —NR^(d1)C(O)NR^(b1)R^(c1),—NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), or —NR^(d1)S(O)₂NR^(b1)R^(c1);e.g., —NR^(b1)R^(c1) or —⁺NR^(b1)R^(c1)R^(d1); e.g., —NH₂, —⁺NH₃, orNHR^(c1).

In some embodiments, X¹ is H, —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g.,CF₃), halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1),—C(O)OH, —C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1),—S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1) (in certainembodiments, X¹ is other than H; e.g., X¹ is —OH, —OR^(a1), —SH,—SR^(a1), —OC(O)H, —OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1),—C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1),—S(O)R^(a1), —S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1)).

In certain embodiments, X¹ is H, —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g.,CF₃), halo (e.g., F), —CN, or —S(O)₂R^(a1) (in certain embodiments, X¹is other than H; e.g., X¹ is —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g.,CF₃), halo (e.g., F), —CN, or —S(O)₂R^(a1)).

In certain embodiments, X¹ is —F, —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1),or —OC(O)NR^(b1)R^(c1). For example, X¹ can be —F, —OH, or —OR^(a1)(e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). As another example,X¹ can be —F or —OH.

In certain embodiments, X¹ is —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1).

In certain embodiments, X¹ is —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1). For example, X¹ can be —OH or —OR^(a1) (e.g.,R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). As another example, X¹ canbe —OH.

In certain embodiments, X¹ is H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH,—C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1),—S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1) (in certainembodiments, X¹ is other than H; e.g., X¹ is C₁₋₄ alkyl, C₁₋₄ haloalkyl(e.g., CF₃), halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1),—C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1),—S(O)R^(a1), —S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1)).

In certain embodiments, X¹ is H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),halo (e.g., F), —CN, or —S(O)₂R^(a1).

In certain embodiments, X¹ is C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),halo (e.g., F), —CN, or —S(O)₂R^(a1).

In certain embodiments, X¹ is H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),or halo (e.g., F).

In certain embodiments, X¹ is C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), orhalo (e.g., F).

In certain embodiments, X¹ is H or halo (e.g., F).

In certain embodiments, X¹ is halo (e.g., —F).

In certain of the foregoing embodiments, two of X², X³, X⁴ and X⁶ (e.g.,X² and X⁴; or X³ and X⁶) c N—R³ (e.g., N—H). In certain of the foregoingembodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³ and X⁶)are N—R³ (e.g., N—H), and the others are O.

In some embodiments, X¹ is —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g.,CF₃), halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1),—C(O)OH, —C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1),—S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1).

In certain embodiments, X¹ is —OH, —OR^(a1), C₁₋₄ alkyl, C₁₋₄ haloalkyl(e.g., CF₃), halo (e.g., F), —CN, and —S(O)₂R^(a1).

In certain embodiments, X¹ is —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1). For example, X¹ can be —OH,—OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1); e.g., X¹ can be—OH or —OR^(a1) (e.g., —OH).

In other embodiments, X¹ is C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo(e.g., F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH,—C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1),—S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1). For example,X¹ can be C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN,or —S(O)₂R^(a1). As another example, X¹ can be C₁₋₄ alkyl, C₁₋₄haloalkyl (e.g., CF₃), or halo (e.g., F). As a further example, X¹ canbe halo (e.g., —F).

In certain of the foregoing embodiments, two of X², X³, X⁴ and X⁶ (e.g.,X² and X⁴; or X³ and X⁶) c N—R³ (e.g., N—H). In certain of the foregoingembodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³ and X⁶)are N—R³ (e.g., N—H), and the others are O.

In some embodiments, the carbon directly attached to X¹ has the(R)-configuration.

In some embodiments, the carbon directly attached to X¹ has the(S)-configuration.

In some embodiments, X⁵ is —OH, —OR^(a1), —F, —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), —NO₂, —N₃,—NR^(d1)C(═NR^(e1))NR^(b1)R^(c1), —NR^(b1)R^(c1), —⁺NR^(b1)R^(c1)R^(d1),—NR^(d1)C(O)H, —NR^(d1)C(O)R^(a1), —NR^(d1)C(O)OR^(a1),—NR^(d1)C(O)NR^(b1)R^(c1), —NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), or—NR^(d1)S(O)₂NR^(b1)R^(c1) (in certain embodiment, X⁵ is other than —F).

In certain embodiments, X⁵ is —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1),—OC(O)NR^(b1)R^(c1), —F, —NO₂, —N₃, —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1),—NR^(b1)R^(c1), —⁺NR^(b1)R^(c1)R^(d1), —NR^(d1)C(O)H,—NR^(d1)C(O)R^(a1), —NR^(d1)C(O)OR^(a1), —NR^(d1)C(O)NR^(b1)R^(c1),—NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), or —NR^(d1)S(O)₂NR^(b1)R^(c1)(in certain embodiment, X⁵ is other than —F).

In certain embodiments, X⁵ is —F, —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1),or —OC(O)NR^(b1)R^(c1) (in certain embodiment, X⁵ is other than —F).

In certain embodiments, X⁵ is —F, —OH or —OR^(a1) (in certainembodiment, X⁵ is other than —F).

In certain embodiments, X⁵ is —OH.

In certain embodiments, X⁵ is —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1) (e.g., —OH or —OR^(a1); e.g., —OH), and two of X²,X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³ and X⁶) are N—R³ (e.g., N—H). Incertain embodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³and X⁶) are N—R³ (e.g., N—H), and the others are O.

In other embodiments, X⁵ is —NO₂, —N₃, —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1),—NR^(b1)R^(c1), —⁺NR^(b1)R^(c1)R^(d1), —NR^(d1)C(O)H,—NR^(d1)C(O)R^(a1), —NR^(d1)C(O)OR^(a1), —NR^(d1)C(O)NR^(b1)R^(c1),—NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), or —NR^(d1)S(O)₂NR^(b1)R^(c1);e.g., —NR^(b1)R^(c1) or —⁺NR^(b1)R^(c1)R^(d1); e.g., —NH₂, —⁺NH₃, orNHR^(c1).

In some embodiments, X⁵ is H, —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g.,CF₃), halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1),—C(O)OH, —C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1),—S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1) (in certainembodiments, X¹ is other than H; e.g., X⁵ is —OH, —OR^(a1), —SH,—SR^(a1), —OC(O)H, —OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1),—C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1),—S(O)R^(a1), —S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1)).

In certain embodiments, X⁵ is H, —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g.,CF₃), halo (e.g., F), —CN, or —S(O)₂R^(a1) (in certain embodiments, X⁵is other than H).

In certain embodiments, X⁵ is —F, —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1),or —OC(O)NR^(b1)R^(c1). For example, X⁵ can be —F, —OH, or —OR^(a1)(e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). As another example,X⁵ can be —F or —OH.

In certain embodiments, X⁵ is —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1).

In certain embodiments, X⁵ is —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1). For example, X⁵ can be —OH or —OR^(a1) (e.g.,R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). As another example, X⁵ canbe —OH.

In certain embodiments, X⁵ is H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH,—C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1),—S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1) (in certainembodiments, X⁵ is other than H; e.g., X⁵ is C₁₋₄ alkyl, C₁₋₄ haloalkyl(e.g., CF₃), halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1),—C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1),—S(O)R^(a1), —S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1)).

In certain embodiments, X⁵ is H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),halo (e.g., F), —CN, or —S(O)₂R^(a1).

In certain embodiments, X⁵ is C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),halo (e.g., F), —CN, or —S(O)₂R^(a1).

In certain embodiments, X⁵ is H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),or halo (e.g., F).

In certain embodiments, X⁵ is C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), orhalo (e.g., F).

In certain embodiments, X⁵ is H or halo (e.g., F).

In certain embodiments, X⁵ is halo (e.g., —F).

In certain of the foregoing embodiments, two of X², X³, X⁴ and X⁶ (e.g.,X² and X⁴; or X³ and X⁶) c N—R³ (e.g., N—H). In certain of the foregoingembodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³ and X⁶)are N—R³ (e.g., N—H), and the others are O.

In some embodiments, X⁵ is —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g.,CF₃), halo (e.g., F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1),—C(O)OH, —C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1),—S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1).

In certain embodiments, X⁵ is —OH, —OR^(a1), C₁₋₄ alkyl, C₁₋₄ haloalkyl(e.g., CF₃), halo (e.g., F), —CN, and —S(O)₂R^(a1).

In certain embodiments, X⁵ is —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H,—OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1). For example, X⁵ can be —OH,—OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1); e.g., X¹ can be—OH or —OR^(a1) (e.g., —OH).

In other embodiments, X⁵ is C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo(e.g., F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH,—C(O)OR^(a1), —C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1),—S(O)NR^(b1)R^(c1), —S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1). For example,X⁵ can be C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN,or —S(O)₂R^(a1). As another example, X⁵ can be C₁₋₄ alkyl, C₁₋₄haloalkyl (e.g., CF₃), or halo (e.g., F). As a further example, X⁵ canbe halo (e.g., —F).

In certain of the foregoing embodiments, two of X², X³, X⁴ and X⁶ (e.g.,X² and X⁴; or X³ and X⁶) are N—R³ (e.g., N—H). In certain of theforegoing embodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³and X⁶) are N—R³ (e.g., N—H), and the others are O.

In some embodiments, the carbon directly attached to X⁵ has the(R)-configuration.

In some embodiments, the carbon directly attached to X⁵ has the(S)-configuration.

In some embodiments, X¹ and X⁵ are each independently selected from —OH,—OR^(a1), —F, —SH, —SR^(a1), —OC(O)H, —OC(O)R^(a1), —OC(O)NR^(b1)R^(c1),—NO₂, —N₃, —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1), —NR^(b1)R^(c1),—+NR^(b1)R^(c1)R^(d1), —NR^(d1)C(O)H, —NR^(d1)C(O)R^(a1),—NR^(d1)C(O)OR^(a1), —NR^(d1)C(O)NR^(b1)R^(c1), —NR^(d1)S(O)R^(a1),—NR^(d1)S(O)₂R^(a1), or —NR^(d1)S(O)₂NR^(b1)R^(c1) (in certainembodiment, X¹ and/or X⁵ is other than —F). X¹ and X⁵ can be the same ordifferent.

In certain embodiments, X¹ and X⁵ are each independently selected from—OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), —OC(O)NR^(b1)R^(c1), —F, —NO₂,—N₃, —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1), —NR^(b1)R^(c1),—⁺NR^(b1)R^(c1)R^(d1), —NR^(d1)C(O)H, —NR^(d1)C(O)R^(a1),—NR^(d1)C(O)OR^(a1), —NR^(d1)C(O)NR^(b1)R^(c1), —NR^(d1)S(O)R^(a1),—NR^(d1)S(O)₂R^(a1), or —NR^(d1)S(O)₂NR^(b1)R^(c1) (in certainembodiment, X¹ and X⁵ are each other than —F).

In certain embodiments, X¹ and X⁵ are each independently selected from—F, —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1) (incertain embodiment, X¹ and X⁵ are each other than —F).

In certain embodiments, X¹ and X⁵ are each independently selected from—F, —OH or —OR^(a1) (in certain embodiment, X¹ and X⁵ are each otherthan —F).

In certain embodiments, X¹ and X⁵ are each —OH.

In certain embodiments, X¹ and X⁵ are each independently selected from—OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1) (e.g., —OHor —OR^(a1); e.g., —OH), and at least one (e.g., two) of X², X³, X⁴ andX⁶ is other than O (e.g., N—R³). In certain embodiments, two of X², X³,X⁴ and X⁶ (e.g., X² and X⁴) are N—R³ (e.g., N—H), and the others are O.

In other embodiments, X¹ and X⁵ are each independently selected from—NO₂, —N₃, —NR^(d1)C(═NR^(e1))NR^(b1)R^(c1), —NR^(b1)R^(c1),—⁺NR^(b1)R^(d1), —NR^(d1)C(O)H, —NR^(d1)C(O)R^(a1), —NR^(d1)C(O)OR^(a1),—NR^(d1)C(O)NR^(b1)R^(c1), —NR^(d1)S(O)R^(a1), —NR^(d1)S(O)₂R^(a1), or—NR^(d1)S(O)₂NR^(b1)R^(c1); e.g., —NR^(b1)R^(c1) or—⁺NR^(b1)R^(c1)R^(d1); e.g., —NH₂, —⁺NH₃, or NHR^(c1).

In some embodiments, X¹ and X⁵ are each independently selected from H,—OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H, —OC(O)R^(a1),—OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g.,F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1) (in certain embodiments, X¹ and X⁵are each other than H; e.g., X¹ and X⁵ are each independently selectedfrom —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H, —OC(O)R^(a1),—OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g.,F), —CN, —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1)).

In certain embodiments, X¹ and X⁵ are each independently selected fromH, —OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H, —OC(O)R^(a1),—OC(O)NR^(b1)R^(c1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g.,F), —CN, and —S(O)₂R^(a1) (in certain embodiments, X¹ and X⁵ are eachother than H; e.g., X¹ and X⁵ are each independently selected from —OH,—OR^(a1), —SH, —SR^(a1), —OC(O)H, —OC(O)R^(a1), —OC(O)NR^(b1)R^(c1),C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1)).

In certain embodiments, X¹ and X⁵ are each independently selected from—F, —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), and —OC(O)NR^(b1)R^(c1). Forexample, X¹ and X⁵ are each independently selected from —F, —OH, and—OR^(a1) (e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). As anotherexample, X¹ and X⁵ are each independently selected from —F and —OH.

In certain embodiments, X¹ and X⁵ are each independently selected from—OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H, —OC(O)R^(a1), and—OC(O)NR^(b1)R^(c1).

In certain embodiments, X¹ and X⁵ are each independently selected from—OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), and —OC(O)NR^(b1)R^(c1). Forexample, X¹ and X⁵ are each independently selected from —OH and —OR^(a1)(e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). As another example,X¹ and X⁵ are each —OH.

In certain embodiments, X¹ and X⁵ are each independently selected fromH, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1) (in certain embodiments, X¹ and X⁵are each other than H; e.g., X¹ and X⁵ are each independently selectedfrom C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN,—C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1)).

In certain embodiments, X¹ and X⁵ are each independently selected fromH, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1).

In certain embodiments, X¹ and X⁵ are each independently selected fromC₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1).

In certain embodiments, X¹ and X⁵ are each independently selected fromH, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), and halo (e.g., F).

In certain embodiments, X¹ and X⁵ are each independently selected fromC₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), and halo (e.g., F).

In certain embodiments, X¹ and X⁵ are each independently selected from Hand halo (e.g., F).

In certain embodiments, X¹ and X⁵ are each an independently selectedhalo (e.g., —F).

In certain of the foregoing embodiments, two of X², X³, X⁴ and X⁶ (e.g.,X² and X⁴; or X³ and X⁶) c N—R³ (e.g., N—H). In certain of the foregoingembodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³ and X⁶)are N—R³ (e.g., N—H), and the others are O.

In some embodiments, X¹ and X⁵ are each independently selected from —OH,—OR^(a1), —SH, —SR^(a1), —OC(O)H, —OC(O)R^(a1), —OC(O)NR^(b1)R^(c1),C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1).

In certain embodiments, X¹ and X⁵ are each independently selected from—OH, —OR^(a1), C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F),—CN, and —S(O)₂R^(a1).

In certain embodiments, X¹ and X⁵ are each independently selected from—OH, —OR^(a1), —SH, —SR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1). For example, X¹ and X⁵ can each be independentlyselected from —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1); e.g., X¹ and X⁵ can each be independently selectedfrom —OH or —OR^(a1) (e.g., —OH).

In other embodiments, X¹ and X⁵ are each independently selected fromC₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), or —S(O)₂NR^(b1)R^(c1). For example, X¹ and X⁵ can each beindependently selected from C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo(e.g., F), —CN, or —S(O)₂R^(a1). As another example, X¹ and X⁵ can eachbe independently selected from C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),or halo (e.g., F). As a further example, X¹ and X⁵ can each be halo(e.g., —F).

In certain of the foregoing embodiments, two of X², X³, X⁴ and X⁶ (e.g.,X² and X⁴; or X³ and X⁶) are N—R³ (e.g., N—H). In certain of theforegoing embodiments, two of X², X³, X⁴ and X⁶ (e.g., X² and X⁴; or X³and X⁶) are N—R³ (e.g., N—H), and the others are O.

In some of the foregoing embodiments, X¹ and X⁵ are the same (e.g., X¹and X⁵ are both —OH; or X¹ and X⁵ are both halo (e.g., X¹ and X⁵ areboth —F); or X¹ and X⁵ are both —OR^(a1), in which R^(a1) can be C₁₋₁₀alkyl, e.g., C₁₋₄ alkyl).

In some of the foregoing embodiments, X¹ and X⁵ are different (e.g., oneof X¹ and X⁵ is —OH, and the other is halo (e.g., —F); or one of X¹ andX⁵ is —OH, and the other is —OR^(a1), in which R^(a1) can be C₁₋₁₀alkyl, e.g., C₁₋₄ alkyl; or one of X¹ and X⁵ is —OR^(a1), in whichR^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl, and the other is halo(e.g., —F)).

In some embodiments, the carbon directly attached to X¹ and the carbondirectly attached to X⁵ both have the (R)-configuration.

In some embodiments, the carbon directly attached to X¹ and the carbondirectly attached to X⁵ both have the (S)-configuration.

In some embodiments, the carbon directly attached to X¹ and the carbondirectly attached to X⁵ have opposite configurations (i.e., one has the(R)-configuration, and the other has the (S)-configuration).

Variables L¹ and L²

In some embodiments, L¹ is

In some embodiments, Y¹ is —OH, —OR^(a1), O⁻, —SH, —SR^(a1), or S. Incertain embodiments, Y¹ is —OH, —OR^(a1), or O⁻ (e.g., —OR^(a1) or O⁻).In other embodiments, Y¹ is S⁻. In certain of these embodiments, L¹ hasthe R_(P) configuration, or L¹ has the S_(P) configuration.

In some embodiments, L² is

In some embodiments, Y² is —OH, —OR^(a1), O⁻, —SH, —SR^(a1), or —S⁻. Incertain embodiments, ⋅Y² is —OH, —OR^(a1), or O⁻ (e.g., —OR^(a1) or O⁻).In other embodiments, Y² is —SH or —S⁻. In certain of these embodiments,L² has the R_(P) configuration, or L² has the S_(P) configuration.

In some embodiments, L¹ is

and L² is

Y¹ and Y² can be the same or different. In some embodiments, Y¹ and Y²are each independently selected from is —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; e.g., —OR^(a1) or O⁻; e.g., —SH or S⁻, e.g., —S⁻.

In certain embodiments, Y¹ and Y² are each —O⁻.

In certain embodiments, Y¹ and Y² are each —SH or —S⁻, e.g., —S⁻. Incertain of these embodiments, L¹ and L² both have the R_(P)configuration or both have the S_(P) configuration. In other of theseembodiments, one of L¹ and L² has the R_(P) configuration, and the otherhas the S_(P) configuration.

Variables R^(1A) and R^(1B) and R^(2A) and R^(2B)

In some embodiments, R^(1A) and R^(1B) are each H. In some embodiments,R^(2A) and R^(2B) are each H. In some embodiments, R^(1A) and R^(1B) areeach H, and R^(2A) and R^(2B) are each H.

Variables R³, R^(b1), R^(c1), R^(d1), and R^(e1)

In some embodiments, each occurrence of R³, R^(b1), R^(c1), R^(d1), andR^(e1) (or each occurrence of R^(b1) and R^(c1); or each occurrence ofR^(c1) and R^(d1); or each occurrence of R³; or each occurrence ofR^(e1)) is independently selected from the group consisting of: H;R^(a1); —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OR^(a1), —OC(O)H,—S(O)R^(a1), and —S(O)₂R^(a1); or

R^(b1) and R^(c1) taken together with the nitrogen atom to which each isattached form a heterocyclyl, including from 3-10 (e.g., 3-6, 4-6, 5-6)ring atoms, wherein from 1-3 ring atoms are independently selected fromthe group consisting of nitrogen, oxygen and sulfur, and which isoptionally substituted with from 1-5 R^(C); (e.g., azetidinyl,morpholino, piperidinyl).

In certain embodiments, each occurrence of R³, R^(b1), R^(c1), R^(d1),and R^(e1) (or each occurrence of R^(b1) and R^(c1); or each occurrenceof R^(c1) and R^(d1); or each occurrence of R³; or each occurrence ofR^(e1)) is independently selected from the group consisting of: H;R^(a1); —C(O)H, —C(O)R^(a1), —C(O)NRR′, wherein R and R′ are eachindependently selected from H and C₁₋₄ alkyl optionally substituted withfrom 1-3 R^(A); —C(O)OR^(a1), —OC(O)H, —S(O)R^(a1), and —S(O)₂R^(a1); or

R^(b1) and R^(c1) taken together with the nitrogen atom to which each isattached form a heterocyclyl, including from 3-10 (e.g., 3-6, 4-6, 5-6)ring atoms, wherein from 1-3 ring atoms are independently selected fromthe group consisting of nitrogen, oxygen and sulfur, and which isoptionally substituted with from 1-5 R^(C); (e.g., azetidinyl,morpholino, piperidinyl).

In certain embodiments, each occurrence of R³, R^(b1), R^(c1), R^(d1),and R^(e) (or each occurrence of R^(b1) and R^(e1); or each occurrenceof R^(c1) and R^(d1); or each occurrence of R³; or each occurrence ofR^(e1)) is independently selected from the group consisting of: H; C₁₋₆(e.g., C₁₋₄) alkyl optionally substituted with from 1-3 R^(A); —SO₂(C₁₋₆alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl optionally substituted with from 1-3R^(A)); —C(O)NRR′, wherein R and R′ are each independently selected fromH and C₁₋₄ alkyl optionally substituted with from 1-3 R^(A); and—C(O)O(C₁₋₆ alkyl optionally substituted with from 1-3 R^(A)); or

R^(b1) and R^(c1) taken together with the nitrogen atom to which each isattached form a heterocyclyl, including from 3-10 (e.g., 3-6, 4-6, 5-6)ring atoms, wherein from 1-3 ring atoms are independently selected fromthe group consisting of nitrogen, oxygen and sulfur, and which isoptionally substituted with from 1-5 R^(C); (e.g., azetidinyl,morpholino, piperidinyl).

In certain embodiments, each occurrence of R³, R^(b1), R^(c1), R^(d1),and R^(e1) (or each occurrence of R^(b1) and R^(c1); or each occurrenceof R^(c1) and R^(d1); or each occurrence of R³; or each occurrence ofR^(e1)) is independently selected from the group consisting of: H; C₁₋₆(e.g., C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl);—C(O)NRR′, wherein R and R′ are each independently selected from H andC₁₋₄ alkyl; and —C(O)O(C₁₋₆ alkyl); or

R^(b1) and R^(c1) taken together with the nitrogen atom to which each isattached form a heterocyclyl, including from 3-10 (e.g., 3-6, 4-6, 5-6)ring atoms, wherein from 1-3 ring atoms are independently selected fromthe group consisting of nitrogen, oxygen and sulfur, and which isoptionally substituted with from 1-5 R^(C); (e.g., azetidinyl,morpholino, piperidinyl).

In certain embodiments, each occurrence of R³, R^(b1), R^(c1), R^(d1),and R^(e1) (or each occurrence of R^(b1) and R^(c1); or each occurrenceof R^(c1) and R^(d1); or each occurrence of R³; or each occurrence ofR^(e1)) is independently selected from the group consisting of: H;R^(a1); —C(O)H, —C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OR^(a1), —OC(O)H,—S(O)R^(a1), and —S(O)₂R^(a1).

In certain embodiments, each occurrence of R³, R^(b1), R^(c1), R^(d1),and R^(e1) (or each occurrence of R^(b1) and R^(c1); or each occurrenceof R^(c1) and R^(d1); or each occurrence of R³; or each occurrence ofR^(e1)) is independently selected from the group consisting of: H;R^(a1); —C(O)H, —C(O)R^(a1), —C(O)NRR′, wherein R and R′ are eachindependently selected from H and C₁₋₄ alkyl optionally substituted withfrom 1-3 R^(A); —C(O)OR^(a1), —OC(O)H, —S(O)R^(a1), and —S(O)₂R^(a1).

In certain embodiments, each occurrence of R³, R^(b1), R^(c1), R^(d1),and R^(e1) (or each occurrence of R^(b1) and R^(c1); or each occurrenceof R^(c1) and R^(d1); or each occurrence of R³; or each occurrence ofR^(e1)) is independently selected from the group consisting of: H; C₁₋₆(e.g., C₁₋₄) alkyl optionally substituted with from 1-3 R^(A); —SO₂(C₁₋₆alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl optionally substituted with from 1-3R^(A)); —C(O)NRR′, wherein R and R′ are each independently selected fromH and C₁₋₄ alkyl optionally substituted with from 1-3 R^(A); and—C(O)O(C₁₋₆ alkyl optionally substituted with from 1-3 R^(A)).

In certain embodiments, each occurrence of R³, R^(b1), R^(c1), R^(d1),and R^(e1) (or each occurrence of R^(b1) and R^(c1); or each occurrenceof R^(c1) and R^(d1); or each occurrence of R³; or each occurrence ofR^(e1)) is independently selected from the group consisting of: H; C₁₋₆(e.g., C₁-4) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl);—C(O)NRR′, wherein R and R′ are each independently selected from H andC₁₋₄ alkyl; and —C(O)O(C₁₋₆ alkyl).

In certain embodiments, R^(b1) and R^(c1) taken together with thenitrogen atom to which each is attached form a heterocyclyl, includingfrom 3-10 (e.g., 3-6, 4-6, 5-6) ring atoms, wherein from 1-3 ring atomsare independently selected from the group consisting of nitrogen, oxygenand sulfur, and which is optionally substituted with from 1-5 R^(C);(e.g., azetidinyl, morpholino, piperidinyl).

Non-Limiting Combinations

[A] In some embodiments:

X¹ and X⁵ are each independently selected from the group consisting of—OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1);

two of X², X³, X⁴ and X⁶ are N—R³ (e.g., N—H);

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:formula (i) and formula (ii).

Embodiments can include any one or more of the features delineated inclaims 83-96 and those delineated below.

Embodiments can include any one or more of the following features.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be N, and Z³ can be N—R³ (e.g., N—H). R^(6′) canbe —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

X¹ and X⁵ can each be independently selected from the group consistingof —OH, or —OR^(a1) (e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl);e.g., X¹ and X⁵ can each be —OH. Two of X², X³, X⁴ and X⁶ can be N—R³(e.g., N—H), and the others can be O. Y¹ and Y² can each be O⁻; or Y¹and Y² can each be SH or S⁻. L¹ and L² can both have the R_(P)configuration or both have the S_(P) configuration; or one of L¹ and L²can have the R_(P) configuration, and the other can have the S_(P)configuration. R^(1A) and R^(1B) can each be H, and R^(2A) and R^(2B)can each be H.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[B] In some embodiments:

X¹ and X⁵ are each independently selected from the group consisting ofH, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1);

two of X², X³, X⁴ and X⁶ are N—R³ (e.g., N—H);

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:

Embodiments can include any one or more of the features delineated inclaims 128-143 and those delineated below.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be. N, and Z³ can be N—R³ (e.g., N—H). R^(6′)can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

X¹ and X⁵ can each be independently selected from the group consistingof H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1) (in certain embodiments, each of X¹ and X⁵ is other than H;e.g., X¹ and X⁵ can each be independently selected from the groupconsisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F),—CN, and —S(O)₂R^(a1)).

In certain embodiments, X¹ and X⁵ can each be independently selectedfrom the group consisting of H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),and halo (e.g., F) (in certain embodiments, each of X¹ and X⁵ is otherthan H; e.g., X¹ and X⁵ can each be independently selected from thegroup consisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), and halo(e.g., F)).

For example, X¹ and X⁵ can each be independently selected from the groupconsisting of H and halo (e.g., F); or X¹ and X⁵ can each be anindependently selected halo (e.g., F).

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[C] In some embodiments, the compound has formula (II):

Embodiments can include any one or more of the features delineatedthroughout the specification, e.g., those delineated in claims 19-81.

[D] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting of—OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1);

two of X², X³, X⁴ and X⁶ are N—R³ (e.g., N—H);

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 83-96 and those delineated below.

X¹ and X⁵ can each be independently selected from the group consistingof —OH, or —OR^(a1) (e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl);e.g., X¹ and X⁵ can each be —OH. Two of X², X³, X⁴ and X⁶ can be N—R³(e.g., N—H), and the others can be O. Y¹ and Y² can each be O⁻; or Y¹and Y² can each be SH or S⁻. L¹ and L² can both have the R_(P)configuration or both have the S_(P) configuration; or one of L¹ and L²can have the R_(P) configuration, and the other can have the S_(P)configuration. R^(1A) and R^(1B) can each be H, and R^(2A) and R^(2B)can each be H.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[E] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting ofH, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1);

two of X², X³, X⁴ and X⁶ are N—R³ (e.g., N—H);

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 128-143 and those delineated below.

X¹ and X⁵ can each be independently selected from the group consistingof H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1) (in certain embodiments, each of X¹ and X⁵ is other than H;e.g., X¹ and X⁵ can each be independently selected from the groupconsisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F),—CN, and —S(O)₂R^(a1)).

In certain embodiments, X¹ and X⁵ can each be independently selectedfrom the group consisting of H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),and halo (e.g., F) (in certain embodiments, each of X¹ and X⁵ is otherthan H; e.g., X¹ and X⁵ can each be independently selected from thegroup consisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), and halo(e.g., F)).

For example, X¹ and X⁵ can each be independently selected from the groupconsisting of H and halo (e.g., F); or X¹ and X⁵ can each be anindependently selected halo (e.g., F).

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[F] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting ofis —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1);

X² and X⁴ are each an independently selected N—R³ (e.g., N—H);

X³ and X⁶ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 98-111 and those delineated below.

X¹ and X⁵ can each be independently selected from the group consistingof —OH, or —OR^(a1) (e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl);e.g., X¹ and X⁵ can each be —OH. Two of X², X³, X⁴ and X⁶ can be N—R³(e.g., N—H), and the others can be O. Y¹ and Y² can each be O⁻; or Y¹and Y² can each be SH or S⁻. L¹ and L² can both have the R_(P)configuration or both have the S_(P) configuration; or one of L¹ and L²can have the R_(P) configuration, and the other can have the S_(P)configuration. R^(1A) and R^(1B) can each be H, and R^(2A) and R^(2B)can each be H.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[G] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting of—OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1);

X³ and X⁶ are each an independently selected N—R³ (e.g., N—H);

X² and X⁴ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 113-126 and those delineated below.

X¹ and X⁵ can each be independently selected from the group consistingof —OH, or —OR^(a1) (e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl);e.g., X¹ and X⁵ can each be —OH. Two of X², X³, X⁴ and X⁶ can be N—R³(e.g., N—H), and the others can be O. Y¹ and Y² can each be O⁻; or Y¹and Y² can each be SH or S⁻. L¹ and L² can both have the R_(P)configuration or both have the S_(P) configuration; or one of L¹ and L²can have the R_(P) configuration, and the other can have the S_(P)configuration. R^(1A) and R^(1B) can each be H, and R^(2A) and R^(2B)can each be H.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[H] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting ofH, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1);

X² and X⁴ are each an independently selected N—R³ (e.g., N—H);

X³ and X⁶ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 145-159 and those delineated below.

X¹ and X⁵ can each be independently selected from the group consistingof H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1) (in certain embodiments, each of X¹ and X⁵ is other than H;e.g., X¹ and X⁵ can each be independently selected from the groupconsisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F),—CN, and —S(O)₂R^(a1)).

In certain embodiments, X¹ and X⁵ can each be independently selectedfrom the group consisting of H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),and halo (e.g., F) (in certain embodiments, each of X¹ and X⁵ is otherthan H; e.g., X¹ and X⁵ can each be independently selected from thegroup consisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), and halo(e.g., F)).

For example, X¹ and X⁵ can each be independently selected from the groupconsisting of H and halo (e.g., F); or X¹ and X⁵ can each be anindependently selected halo (e.g., F).

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[I] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting ofis are each independently selected from the group consisting of H, C₁₋₄alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1);

X³ and X⁶ are each an independently selected N—R³ (e.g., N—H);

X² and X⁴ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 161-175 and those delineated below.

X¹ and X⁵ can each be independently selected from the group consistingof H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1) (in certain embodiments, each of X¹ and X⁵ is other than H;e.g., X¹ and X⁵ can each be independently selected from the groupconsisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F),—CN, and —S(O)₂R^(a1)).

In certain embodiments, X¹ and X⁵ can each be independently selectedfrom the group consisting of H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),and halo (e.g., F) (in certain embodiments, each of X¹ and X⁵ is otherthan H; e.g., X¹ and X⁵ can each be independently selected from thegroup consisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), and halo(e.g., F)).

For example, X¹ and X⁵ can each be independently selected from the groupconsisting of H and halo (e.g., F); or X¹ and X⁵ can each be anindependently selected halo (e.g., F).

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[J] In some embodiments, the compound has formula (VI):

in which,

X¹ and X⁵ are each independently selected from the group consisting ofis —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1);

X²¹ and X⁴¹ are each an independently selected N—R³ (e.g., N—H);

X³¹ and X⁶¹ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:formula (i) and formula (ii).

Embodiments can include any one or more of the features delineated inclaims 97-111 and those delineated below.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be. N, and Z³ can be N—R³ (e.g., N—H). R^(6′)can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

X¹ and X⁵ can each be independently selected from the group consistingof —OH, or —OR^(a1) (e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl);e.g., X¹ and X⁵ can each be —OH. Two of X², X³, X⁴ and X⁶ can be N—R³(e.g., N—H), and the others can be O. Y¹ and Y² can each be O⁻; or Y¹and Y² can each be SH or S⁻. L¹ and L² can both have the R_(P)configuration or both have the S_(P) configuration; or one of L¹ and L²can have the R_(P) configuration, and the other can have the SEconfiguration. R^(1A) and R^(1B) can each be H, and R^(2A) and R^(2B)can each be H.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[K] In some embodiments, the compound has formula (VI), in which:

X¹ and X⁵ are each independently selected from the group consisting of—OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or —OC(O)NR^(b1)R^(c1);

X³¹ and X⁶¹ are each an independently selected N—R³ (e.g., N—H);

X²¹ and X⁴¹ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:

Embodiments can include any one or more of the features delineated inclaims 113-126 and those delineated below.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be N, and Z³ can be N—R³ (e.g., N—H). R^(6′) canbe —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

X¹ and X⁵ can each be independently selected from the group consistingof —OH, or —OR^(a1) (e.g., R^(a1) can be C₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl);e.g., X¹ and X⁵ can each be —OH. Two of X², X³, X⁴ and X⁶ can be N—R³(e.g., N—H), and the others can be O. Y¹ and Y² can each be O⁻; or Y andY² can each be SH or S⁻. L¹ and L² can both have the R_(P) configurationor both have the S_(P) configuration; or one of L¹ and L² can have theR_(P) configuration, and the other can have the S_(P) configuration.R^(1A) and R^(1B) can each be H, and R^(2A) and R^(2B) can each be H.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[L] In some embodiments, the compound has formula (VI), in which:

wherein,

X¹ and X⁵ are each independently selected from the group consisting ofH, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R^(c1);

X²¹ and X⁴¹ are each an independently selected N—R³ (e.g., N—H);

X³¹ and X⁶¹ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:

Embodiments can include any one or more of the features delineated inclaims 145-159 and those delineated below.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be. N, and Z³ can be N—R³ (e.g., N—H). R^(6′)can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

X¹ and X⁵ can each be independently selected from the group consistingof H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1) (in certain embodiments, each of X¹ and X⁵ is other than H;e.g., X¹ and X⁵ can each be independently selected from the groupconsisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F),—CN, and —S(O)₂R^(a1)).

In certain embodiments, X¹ and X⁵ can each be independently selectedfrom the group consisting of H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),and halo (e.g., F) (in certain embodiments, each of X¹ and X⁵ is otherthan H; e.g., X¹ and X⁵ can each be independently selected from thegroup consisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), and halo(e.g., F)).

For example, X¹ and X⁵ can each be independently selected from the groupconsisting of H and halo (e.g., F); or X¹ and X⁵ can each be anindependently selected halo (e.g., F).

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[M] In some embodiments, the compound has formula (VI), in which:

X¹ and X⁵ are each independently selected from the group consisting ofis are each independently selected from the group consisting of H, C₁₋₄alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, —C(O)H,—C(O)R^(a1), —C(O)NR^(c1)R^(d1), —C(O)OH, —C(O)OR^(a1),—C(═NR^(e1))NR^(b1)R^(c1), —S(O)R^(a1), —S(O)NR^(b1)R^(c1),—S(O)₂R^(a1), and —S(O)₂NR^(b1)R;

X³¹ and X⁶¹ are each an independently selected N—R³ (e.g., N—H);

X²¹ and X⁴¹ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:

Embodiments can include any one or more of the features delineated inclaims 161-175 and those delineated below.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be N, and Z³ can be N—R³ (e.g., N—H). R^(6′) canbe —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

X¹ and X⁵ can each be independently selected from the group consistingof H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F), —CN, and—S(O)₂R^(a1) (in certain embodiments, each of X¹ and X⁵ is other than H;e.g., X¹ and X⁵ can each be independently selected from the groupconsisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), halo (e.g., F),—CN, and —S(O)₂R^(a1)).

In certain embodiments, X¹ and X⁵ can each be independently selectedfrom the group consisting of H, C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃),and halo (e.g., F) (in certain embodiments, each of X¹ and X⁵ is otherthan H; e.g., X¹ and X⁵ can each be independently selected from thegroup consisting of C₁₋₄ alkyl, C₁₋₄ haloalkyl (e.g., CF₃), and halo(e.g., F)).

For example, X¹ and X⁵ can each be independently selected from the groupconsisting of H and halo (e.g., F); or X¹ and X⁵ can each be anindependently selected halo (e.g., F).

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[N] In some embodiments:

X¹ and X⁵ are each independently selected from the group consisting ofhalo (e.g., —F), —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1);

two of X², X³, X⁴ and X⁶ are N—R³ (e.g., N—H);

L¹ 1;

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:formula (i) and formula (ii).

Embodiments can include any one or more of the features delineated inclaims 177-190 and those delineated below.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be N, and Z³ can be N—R³ (e.g., N—H). R^(6′) canbe —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

Each of X¹ and X⁵ can each be independently selected from the groupconsisting of halo (e.g., —F), —OH, and —OR^(a1) (e.g., R^(a1) can beC₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). In certain embodiments, each of X¹ andX⁵ can each be independently selected from the group consisting of: halo(e.g., —F) and —OH. For example, one of X¹ and X⁵ can be halo (e.g.,—F), and the other can be —OH.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[O] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting ofhalo (e.g., —F), —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1);

two of X², X³, X⁴ and X⁶ are N—R³ (e.g., N—H);

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 177-190 and those delineated below.

Each of X¹ and X⁵ can each be independently selected from the groupconsisting of halo (e.g., —F), —OH, and —OR^(a1) (e.g., R^(a1) can beC₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). In certain embodiments, each of X¹ andX⁵ can each be independently selected from the group consisting of: halo(e.g., —F) and —OH. For example, one of X¹ and X⁵ can be halo (e.g.,—F), and the other can be —OH.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[P] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting ofis halo (e.g., —F), —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1);

X² and X⁴ are each an independently selected N—R³ (e.g., N—H);

X³ and X⁶ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 192-204 and those delineated below.

Each of X¹ and X⁵ can each be independently selected from the groupconsisting of halo (e.g., —F), —OH, and —OR^(a1) (e.g., R^(a1) can beC₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). In certain embodiments, each of X¹ andX⁵ can each be independently selected from the group consisting of: halo(e.g., —F) and —OH. For example, one of X¹ and X⁵ can be halo (e.g.,—F), and the other can be —OH.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[Q] In some embodiments of formula (II), (II-A), (III), or (IV):

X¹ and X⁵ are each independently selected from the group consisting ofhalo (e.g., —F), —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1);

X³ and X⁶ are each an independently selected N—R³ (e.g., N—H);

X² and X⁴ are O; L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; and optionally:

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —NR^(b1)R^(c1) (e.g.,—NH₂ or —NHR^(c1)); and in certain of these embodiments, R⁴ and/or R⁶ isH; or R⁴ is other than H, and R⁶ is H; and/or

each occurrence of Z¹ is N, Z^(1′) is N, and R⁵ is —OH; in certain ofthese embodiments, R⁶ is H; in certain of these embodiments, R⁴ is H; inother embodiments, R⁴ is other than H; and/or

each occurrence of Z² is N, Z^(2′) is. N, Z³ is N—R³ (e.g., N—H), andR^(6′) is —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1)); and in certain ofthese embodiments, R^(4′) is H; in other embodiments, R^(4′) is otherthan H.

Embodiments can include any one or more of the features delineated inclaims 206-218 and those delineated below.

Each of X¹ and X⁵ can each be independently selected from the groupconsisting of halo (e.g., —F), —OH, and —OR^(a1) (e.g., R^(a1) can beC₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). In certain embodiments, each of X¹ andX⁵ can each be independently selected from the group consisting of: halo(e.g., —F) and —OH. For example, one of X¹ and X⁵ can be halo (e.g.,—F), and the other can be —OH.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[R] In some embodiments, the compound has formula (VI):

in which,

X¹ and X⁵ are each independently selected from the group consisting ofis halo (e.g., —F), —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1);

X²¹ and X⁴¹ are each an independently selected N—R³ (e.g., N—H);

X³¹ and X⁶¹ are O; L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:formula (i) and formula (ii).

Embodiments can include any one or more of the features delineated inclaims 192-204 and those delineated below.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be N, and Z³ can be N—R³ (e.g., N—H). R^(6′) canbe —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

Each of X¹ and X⁵ can each be independently selected from the groupconsisting of halo (e.g., —F), —OH, and —OR^(a1) (e.g., R^(a1) can beC₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). In certain embodiments, each of X¹ andX⁵ can each be independently selected from the group consisting of: halo(e.g., —F) and —OH. For example, one of X¹ and X⁵ can be halo (e.g.,—F), and the other can be —OH.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

[S] In some embodiments, the compound has formula (VI), in which:

X¹ and X⁵ are each independently selected from the group consisting ofhalo (e.g., —F), —OH, —OR^(a1), —OC(O)H, —OC(O)R^(a1), or—OC(O)NR^(b1)R^(c1);

X³¹ and X⁶¹ are each an independently selected N—R³ (e.g., N—H);

X^(2′) and X⁴¹ are O;

L¹ is

L² is

Y¹ and Y² are each independently selected from —OH, —OR^(a1), O⁻, —SH,—SR^(a1), or S; ⋅and

A and B are each independently selected from the group consisting of:

Embodiments can include any one or more of the features delineated inclaims 206-218 and those delineated below.

A can have formula (i), and B can have formula (ii); or A can haveformula (ii), and B can have formula (ii); or A can have formula (i),and B can have formula (i); or A can have formula (ii), and B can haveformula (i). Z¹ can be N, and Z^(1′) can be N. In certain embodiments,R⁵ can be —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R⁴ and/or R⁶ is H; or R⁴ is other than H, and R⁶ is H). Inother embodiments, R⁵ is —OH, and R⁶ is H (e.g., in certain embodiments,R⁴ is H; in other embodiments, R⁴ is other than H). Each occurrence ofZ² can be N, Z^(2′) can be N, and Z³ can be N—R³ (e.g., N—H). R^(6′) canbe —NR^(b1)R^(c1) (e.g., —NH₂ or —NHR^(c1); e.g., in certainembodiments, R^(4′) is H; in other embodiments, R^(4′) is other than H).

Each of X¹ and X⁵ can each be independently selected from the groupconsisting of halo (e.g., —F), —OH, and —OR^(a1) (e.g., R^(a1) can beC₁₋₁₀ alkyl, e.g., C₁₋₄ alkyl). In certain embodiments, each of X¹ andX⁵ can each be independently selected from the group consisting of: halo(e.g., —F) and —OH. For example, one of X¹ and X⁵ can be halo (e.g.,—F), and the other can be —OH.

Each occurrence of R^(b1) and R^(c1) or each occurrence of R^(c1) can beindependently selected from the group consisting of: H; C₁₋₆ (e.g.,C₁₋₄) alkyl; —SO₂(C₁₋₆ alkyl); —C(O)H; —C(O)(C₁₋₆ alkyl); —C(O)NRR′,wherein R′ and R″ are each independently selected from H and C₁₋₄ alkyl;and —C(O)O(C₁₋₆ alkyl).

Representative and non-limiting examples of formula I compounds areshown in Table 1.

TABLE 1

41

42

43

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

61

62

63

71

72

73

74

75

76

77

78

72-0A1

74-0B0

76-0A1

Pharmaceutical Compositions and Administration

General

In some embodiments, a chemical entity (e.g., a compound that modulates(e.g., agonizes) STING, or a pharmaceutically acceptable salt, and/orhydrate, and/or cocrystal, and/or drug combination thereof) isadministered as a pharmaceutical composition that includes the chemicalentity and one or more pharmaceutically acceptable excipients, andoptionally one or more additional therapeutic agents as describedherein.

In some embodiments, the chemical entities can be administered incombination with one or more conventional pharmaceutical excipients.Pharmaceutically acceptable excipients include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifyingdrug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol1000 succinate, surfactants used in pharmaceutical dosage forms such asTweens, poloxamers or other similar polymeric delivery matrices, serumproteins, such as human serum albumin, buffer substances such asphosphates, tris, glycine, sorbic acid, potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium-chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethyl cellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, andwool fat. Cyclodextrins such as α-, β, and γ-cyclodextrin, or chemicallymodified derivatives such as hydroxyalkylcyclodextrins, including 2- and3-hydroxypropyl-β-cyclodextrins, or other solubilized derivatives canalso be used to enhance delivery of compounds described herein. Dosageforms or compositions containing a chemical entity as described hereinin the range of 0.005% to 100% with the balance made up from non-toxicexcipient may be prepared. The contemplated compositions may contain0.001%-100% of a chemical entity provided herein, in one embodiment0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington: TheScience and Practice of Pharmacy, 22^(nd) Edition (Pharmaceutical Press,London, U K. 2012).

Routes of Administration and Composition Components

In some embodiments, the chemical entities described herein or apharmaceutical composition thereof can be administered to subject inneed thereof by any accepted route of administration. Acceptable routesof administration include, but are not limited to, buccal, cutaneous,endocervical, endosinusial, endotracheal, enteral, epidural,interstitial, intra-abdominal, intra-arterial, intrabronchial,intrabursal, intracerebral, intracisternal, intracoronary, intradermal,intraductal, intraduodenal, intradural, intraepidermal, intraesophageal,intragastric, intragingival, intraileal, intralymphatic, intramedullary,intrameningeal, intramuscular, intraovarian, intraperitoneal,intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial,intratesticular, intrathecal, intratubular, intratumoral, intrauterine,intravascular, intravenous, nasal, nasogastric, oral, parenteral,percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous,sublingual, submucosal, topical, transdermal, transmucosal,transtracheal, ureteral, urethral and vaginal. In certain embodiments, apreferred route of administration is parenteral (e.g., intratumoral).

Compositions can be formulated for parenteral administration, e.g.,formulated for injection via the intravenous, intramuscular,sub-cutaneous, or even intraperitoneal routes. Typically, suchcompositions can be prepared as injectables, either as liquid solutionsor suspensions; solid forms suitable for use to prepare solutions orsuspensions upon the addition of a liquid prior to injection can also beprepared; and the preparations can also be emulsified. The preparationof such formulations will be known to those of skill in the art in lightof the present disclosure.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil, or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that it may be easily injected. It also should be stableunder the conditions of manufacture and storage and must be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi.

The carrier also can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion, and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques, which yield a powder of the active ingredient, plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Intratumoral injections are discussed, e.g., in Lammers, et al., “Effectof Intratumoral Injection on the Biodistribution and the TherapeuticPotential of HPMA Copolymer-Based Drug Delivery Systems” Neoplasia.2006, 10, 788-795.

Pharmacologically acceptable excipients usable in the rectal compositionas a gel, cream, enema, or rectal suppository, include, withoutlimitation, any one or more of cocoa butter glycerides, syntheticpolymers such as polyvinylpyrrolidone, PEG (like PEG ointments),glycerine, glycerinated gelatin, hydrogenated vegetable oils,poloxamers, mixtures of polyethylene glycols of various molecularweights and fatty acid esters of polyethylene glycol Vaseline, anhydrouslanolin, shark liver oil, sodium saccharinate, menthol, sweet almondoil, sorbitol, sodium benzoate, anoxid SBN, vanilla essential oil,aerosol, parabens in phenoxyethanol, sodium methyl p-oxybenzoate, sodiumpropyl p-oxybenzoate, diethylamine, carbomers, carbopol,methyloxybenzoate, macrogol cetostearyl ether, cocoyl caprylocaprate,isopropyl alcohol, propylene glycol, liquid paraffin, xanthan gum,carboxy-metabisulfite, sodium edetate, sodium benzoate, potassiummetabisulfite, grapefruit seed extract, methyl sulfonyl methane (MSM),lactic acid, glycine, vitamins, such as vitamin A and E and potassiumacetate.

In certain embodiments, suppositories can be prepared by mixing thechemical entities described herein with suitable non-irritatingexcipients or carriers such as cocoa butter, polyethylene glycol or asuppository wax which are solid at ambient temperature but liquid atbody temperature and therefore melt in the rectum and release the activecompound. In other embodiments, compositions for rectal administrationare in the form of an enema.

In other embodiments, the compounds described herein or a pharmaceuticalcomposition thereof are suitable for local delivery to the digestive orGI tract by way of oral administration (e.g., solid or liquid dosageforms.).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the chemicalentity is mixed with one or more pharmaceutically acceptable excipients,such as sodium citrate or dicalcium phosphate and/or: a) fillers orextenders such as starches, lactose, sucrose, glucose, mannitol, andsilicic acid, b) binders such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c)humectants such as glycerol, d) disintegrating agents such as agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certainsilicates, and sodium carbonate, e) solution retarding agents such asparaffin, f) absorption accelerators such as quaternary ammoniumcompounds, g) wetting agents such as, for example, cetyl alcohol andglycerol monostearate, h) absorbents such as kaolin and bentonite clay,and i) lubricants such as talc, calcium stearate, magnesium stearate,solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof.In the case of capsules, tablets and pills, the dosage form may alsocomprise buffering agents. Solid compositions of a similar type may alsobe employed as fillers in soft and hard-filled gelatin capsules usingsuch excipients as lactose or milk sugar as well as high molecularweight polyethylene glycols and the like.

In one embodiment, the compositions will take the form of a unit dosageform such as a pill or tablet and thus the composition may contain,along with a chemical entity provided herein, a diluent such as lactose,sucrose, dicalcium phosphate, or the like; a lubricant such as magnesiumstearate or the like; and a binder such as starch, gum acacia,polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or thelike. In another solid dosage form, a powder, marume, solution orsuspension (e.g., in propylene carbonate, vegetable oils, PEG's,poloxamer 124 or triglycerides) is encapsulated in a capsule (gelatin orcellulose base capsule). Unit dosage forms in which one or more chemicalentities provided herein or additional active agents are physicallyseparated are also contemplated; e.g., capsules with granules (ortablets in a capsule) of each drug; two-layer tablets; two-compartmentgel caps, etc. Enteric coated or delayed release oral dosage forms arealso contemplated.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid.

In certain embodiments the excipients are sterile and generally free ofundesirable matter. These compositions can be sterilized byconventional, well-known sterilization techniques. For various oraldosage form excipients such as tablets and capsules sterility is notrequired. The USP/NF standard is usually sufficient.

In certain embodiments, solid oral dosage forms can further include oneor more components that chemically and/or structurally predispose thecomposition for delivery of the chemical entity to the stomach or thelower GI; e.g., the ascending colon and/or transverse colon and/ordistal colon and/or small bowel. Exemplary formulation techniques aredescribed in, e.g., Filipski, K. J., et al., Current Topics in MedicinalChemistry, 2013, 13, 776-802, which is incorporated herein by referencein its entirety.

Examples include upper-GI targeting techniques, e.g., Accordion Pill(Intec Pharma), floating capsules, and materials capable of adhering tomucosal walls.

Other examples include lower-GI targeting techniques. For targetingvarious regions in the intestinal tract, several enteric/pH-responsivecoatings and excipients are available. These materials are typicallypolymers that are designed to dissolve or erode at specific pH ranges,selected based upon the GI region of desired drug release. Thesematerials also function to protect acid labile drugs from gastric fluidor limit exposure in cases where the active ingredient may be irritatingto the upper GI (e.g., hydroxypropyl methylcellulose phthalate series,Coateric (polyvinyl acetate phthalate), cellulose acetate phthalate,hydroxypropyl methylcellulose acetate succinate, Eudragit series(methacrylic acid-methyl methacrylate copolymers), and Marcoat). Othertechniques include dosage forms that respond to local flora in the GItract, Pressure-controlled colon delivery capsule, and Pulsincap.

Ocular compositions can include, without limitation, one or more of anyof the following: viscogens (e.g., Carboxymethylcellulose, Glycerin,Polyvinylpyrrolidone, Polyethylene glycol); Stabilizers (e.g., Pluronic(triblock copolymers), Cyclodextrins); Preservatives (e.g., Benzalkoniumchloride, ETDA, SofZia (boric acid, propylene glycol, sorbitol, and zincchloride; Alcon Laboratories, Inc.), Purite (stabilized oxychlorocomplex; Allergan, Inc.)).

Topical compositions can include ointments and creams. Ointments aresemisolid preparations that are typically based on petrolatum or otherpetroleum derivatives. Creams containing the selected active agent aretypically viscous liquid or semisolid emulsions, often eitheroil-in-water or water-in-oil. Cream bases are typically water-washable,and contain an oil phase, an emulsifier and an aqueous phase. The oilphase, also sometimes called the “internal” phase, is generallycomprised of petrolatum and a fatty alcohol such as cetyl or stearylalcohol; the aqueous phase usually, although not necessarily, exceedsthe oil phase in volume, and generally contains a humectant. Theemulsifier in a cream formulation is generally a nonionic, anionic,cationic or amphoteric surfactant. As with other carriers or vehicles,an ointment base should be inert, stable, nonirritating andnon-sensitizing.

In any of the foregoing embodiments, pharmaceutical compositionsdescribed herein can include one or more one or more of the following:lipids, interbilayer crosslinked multilamellar vesicles, biodegradeablepoly(D,L-lactic-co-glycolic acid) [PLGA]-based or poly anhydride-basednanoparticles or microparticles, and nanoporous particle-supported lipidbilayers.

Dosages

The dosages may be varied depending on the requirement of the patient,the severity of the condition being treating and the particular compoundbeing employed. Determination of the proper dosage for a particularsituation can be determined by one skilled in the medical arts. Thetotal daily dosage may be divided and administered in portionsthroughout the day or by means providing continuous delivery.

In some embodiments, the compounds described herein are administered ata dosage of from about 0.001 mg/Kg to about 500 mg/Kg (e.g., from about0.001 mg/Kg to about 200 mg/Kg; from about 0.01 mg/Kg to about 200mg/Kg; from about 0.01 mg/Kg to about 150 mg/Kg; from about 0.01 mg/Kgto about 100 mg/Kg; from about 0.01 mg/Kg to about 50 mg/Kg; from about0.01 mg/Kg to about 10 mg/Kg; from about 0.01 mg/Kg to about 5 mg/Kg;from about 0.01 mg/Kg to about 1 mg/Kg; from about 0.01 mg/Kg to about0.5 mg/Kg; from about 0.01 mg/Kg to about 0.1 mg/Kg; from about 0.1mg/Kg to about 200 mg/Kg; from about 0.1 mg/Kg to about 150 mg/Kg; fromabout 0.1 mg/Kg to about 100 mg/Kg; from about 0.1 mg/Kg to about 50mg/Kg; from about 0.1 mg/Kg to about 10 mg/Kg; from about 0.1 mg/Kg toabout 5 mg/Kg; from about 0.1 mg/Kg to about 1 mg/Kg; from about 0.1mg/Kg to about 0.5 mg/Kg).

Regimens

The foregoing dosages can be administered on a daily basis (e.g., as asingle dose or as two or more divided doses) or non-daily basis (e.g.,every other day, every two days, every three days, once weekly, twiceweeks, once every two weeks, once a month).

In some embodiments, the period of administration of a compounddescribed herein is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, or more. In a furtherembodiment, a period of during which administration is stopped is for 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, or more. In an embodiment, a therapeutic compound isadministered to an individual for a period of time followed by aseparate period of time. In another embodiment, a therapeutic compoundis administered for a first period and a second period following thefirst period, with administration stopped during the second period,followed by a third period where administration of the therapeuticcompound is started and then a fourth period following the third periodwhere administration is stopped. In an aspect of this embodiment, theperiod of administration of a therapeutic compound followed by a periodwhere administration is stopped is repeated for a determined orundetermined period of time. In a further embodiment, a period ofadministration is for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks,11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9months, 10 months, 11 months, 12 months, or more. In a furtherembodiment, a period of during which administration is stopped is for 1day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 12 months, or more.

Methods of Treatment

In some embodiments, methods for treating a subject having condition,disease or disorder in which a decrease or increase in STING activity(e.g., a decrease, e.g., repressed or impaired STING signaling)contributes to the pathology and/or symptoms and/or progression of thecondition, disease or disorder (e.g., immune disorders, cancer) areprovided. In certain embodiments, the chemical entities described hereininduce an immune response in a subject (e.g., a human). In certainembodiments, the chemical entities described herein induceSTING-dependent type I interferon production in a subject (e.g., ahuman).

Indications

In some embodiments, the condition, disease or disorder is cancer.Non-limiting examples of cancer include melanoma, carcinoma, lymphoma,blastoma, sarcoma, and leukemia or lymphoid malignancies. Moreparticular examples of such cancers include breast cancer, colon cancer,rectal cancer, colorectal cancer, kidney or renal cancer, clear cellcancer lung cancer including small-cell lung cancer, non-small cell lungcancer, adenocarcinoma of the lung and squamous carcinoma of the lung,squamous cell cancer (e.g. epithelial squamous cell cancer), cervicalcancer, ovarian cancer, prostate cancer, prostatic neoplasms, livercancer, bladder cancer, cancer of the peritoneum, hepatocellular cancer,gastric or stomach cancer including gastrointestinal cancer,gastrointestinal stromal tumor, pancreatic cancer, head and neck cancer,glioblastoma, retinoblastoma, astrocytoma, thecomas, arrhenoblastomas,hepatoma, hematologic malignancies including non-Hodgkins lymphoma(NHL), multiple myeloma, myelodysplasia disorders, myeloproliferativedisorders, chronic myelogenous leukemia, and acute hematologicmalignancies, endometrial or uterine carcinoma, endometriosis,endometrial stromal sarcoma, fibrosarcomas, choriocarcinoma, salivarygland carcinoma, vulval cancer, thyroid cancer, esophageal carcinomas,hepatic carcinoma, anal carcinoma, penile carcinoma, nasopharyngealcarcinoma, laryngeal carcinomas, Kaposi's sarcoma, mast cell sarcoma,ovarian sarcoma, uterine sarcoma, melanoma, malignant mesothelioma, skincarcinomas, Schwannoma, oligodendroglioma, neuroblastomas,neuroectodermal tumor, rhabdomyosarcoma, osteogenic sarcoma,leiomyosarcomas, Ewing Sarcoma, peripheral primitive neuroectodermaltumor, urinary tract carcinomas, thyroid carcinomas, Wilm's tumor, aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), and Meigs' syndrome.In some cases, the cancer is melanoma.

In some embodiments, the condition, disease or disorder is aneurological disorder, which includes disorders that involve the centralnervous system (brain, brainstem and cerebellum), the peripheral nervoussystem (including cranial nerves), and the autonomic nervous system(parts of which are located in both central and peripheral nervoussystem). Non-limiting examples of cancer include acquired epileptiformaphasia; acute disseminated encephalomyelitis; adrenoleukodystrophy;age-related macular degeneration; agenesis of the corpus callosum;agnosia; Aicardi syndrome; Alexander disease; Alpers' disease;alternating hemiplegia; Alzheimer's disease; Vascular dementia;amyotrophic lateral sclerosis; anencephaly; Angelman syndrome;angiomatosis; anoxia; aphasia; apraxia; arachnoid cysts; arachnoiditis;Anronl-Chiari malformation; arteriovenous malformation; Aspergersyndrome; ataxia telegiectasia; attention deficit hyperactivitydisorder; autism; autonomic dysfunction; back pain; Batten disease;Behcet's disease; Bell's palsy; benign essential blepharospasm; benignfocal; amyotrophy; benign intracranial hypertension; Binswanger'sdisease; blepharospasm; Bloch Sulzberger syndrome; brachial plexusinjury; brain abscess; brain injury; brain tumors (includingglioblastoma multiforme); spinal tumor; Brown-Sequard syndrome; Canavandisease; carpal tunnel syndrome; causalgia; central pain syndrome;central pontine myelinolysis; cephalic disorder; cerebral aneurysm;cerebral arteriosclerosis; cerebral atrophy; cerebral gigantism;cerebral palsy; Charcot-Marie-Tooth disease; chemotherapy-inducedneuropathy and neuropathic pain; Chiari malformation; chorea; chronicinflammatory demyelinating polyneuropathy; chronic pain; chronicregional pain syndrome; Coffin Lowry syndrome; coma, includingpersistent vegetative state; congenital facial diplegia; corticobasaldegeneration; cranial arteritis; craniosynostosis; Creutzfeldt-Jakobdisease; cumulative trauma disorders; Cushing's syndrome; cytomegalicinclusion body disease; cytomegalovirus infection; dancing eyes-dancingfeet syndrome; Dandy-Walker syndrome; Dawson disease; De Morsier'ssyndrome; Dejerine-Klumke palsy; dementia; dermatomyositis; diabeticneuropathy; diffuse sclerosis; dysautonomia; dysgraphia; dyslexia;dystonias; early infantile epileptic encephalopathy; empty sellasyndrome; encephalitis; encephaloceles; encephalotrigeminalangiomatosis; epilepsy; Erb's palsy; essential tremor; Fabry's disease;Fahr's syndrome; fainting; familial spastic paralysis; febrile seizures;Fisher syndrome; Friedreich's ataxia; fronto-temporal dementia and other“tauopathies”; Gaucher's disease; Gerstmann's syndrome; giant cellarteritis; giant cell inclusion disease; globoid cell leukodystrophy;Guillain-Barre syndrome; HTLV-1-associated myelopathy;Hallervorden-Spatz disease; head injury; headache; hemifacial spasm;hereditary spastic paraplegia; heredopathia atactica polyneuritiformis;herpes zoster oticus; herpes zoster; Hirayama syndrome; HIV-associateddementia and neuropathy (also neurological manifestations of AIDS);holoprosencephaly; Huntington's disease and other polyglutamine repeatdiseases; hydranencephaly; hydrocephalus; hypercortisolism; hypoxia;immune-mediated encephalomyelitis; inclusion body myositis;incontinentia pigmenti; infantile phytanic acid storage disease;infantile refsum disease; infantile spasms; inflammatory myopathy;intracranial cyst; intracranial hypertension; Joubert syndrome;Kearns-Sayre syndrome; Kennedy disease Kinsbourne syndrome; Klippel Feilsyndrome; Krabbe disease; Kugelberg-Welander disease; kuru; Laforadisease; Lambert-Eaton myasthenic syndrome; Landau-Kleffner syndrome;lateral medullary (Wallenberg) syndrome; learning disabilities; Leigh'sdisease; Lennox-Gustaut syndrome; Lesch-Nyhan syndrome; leukodystrophy;Lewy body dementia; Lissencephaly; locked-in syndrome; Lou Gehrig'sdisease (i.e., motor neuron disease or amyotrophic lateral sclerosis);lumbar disc disease; Lyme disease—neurological sequelae; Machado-Josephdisease; macrencephaly; megalencephaly; Melkersson-Rosenthal syndrome;Menieres disease; meningitis; Menkes disease; metachromaticleukodystrophy; microcephaly; migraine; Miller Fisher syndrome;mini-strokes; mitochondrial myopathies; Mobius syndrome; monomelicamyotrophy; motor neuron disease; Moyamoya disease;mucopolysaccharidoses; milti-infarct dementia; multifocal motorneuropathy; multiple sclerosis and other demyelinating disorders;multiple system atrophy with postural hypotension; p muscular dystrophy;myasthenia gravis; myelinoclastic diffuse sclerosis; myoclonicencephalopathy of infants; myoclonus; myopathy; myotonia congenital;narcolepsy; neurofibromatosis; neuroleptic malignant syndrome;neurological manifestations of AIDS; neurological sequelae of lupus;neuromyotonia; neuronal ceroid lipofuscinosis; neuronal migrationdisorders; Niemann-Pick disease; O'Sullivan-McLeod syndrome; occipitalneuralgia; occult spinal dysraphism sequence; Ohtahara syndrome;olivopontocerebellar atrophy; opsoclonus myoclonus; optic neuritis;orthostatic hypotension; overuse syndrome; paresthesia; Parkinson'sdisease; paramyotonia congenital; paraneoplastic diseases; paroxysmalattacks; Parry Romberg syndrome; Pelizaeus-Merzbacher disease; periodicparalyses; peripheral neuropathy; painful neuropathy and neuropathicpain; persistent vegetative state; pervasive developmental disorders;photic sneeze reflex; phytanic acid storage disease; Pick's disease;pinched nerve; pituitary tumors; polymyositis; porencephaly; post-poliosyndrome; postherpetic neuralgia; postinfectious encephalomyelitis;postural hypotension; Prader-Willi syndrome; primary lateral sclerosis;prion diseases; progressive hemifacial atrophy; progressive multifocalleukoencephalopathy; progressive sclerosing poliodystrophy; progressivesupranuclear palsy; pseudotumor cerebri; Ramsay-Hunt syndrome (types Iand II); Rasmussen's encephalitis; reflex sympathetic dystrophysyndrome; Refsum disease; repetitive motion disorders; repetitive stressinjuries; restless legs syndrome; retrovirus-associated myelopathy; Rettsyndrome; Reye's syndrome; Saint Vitus dance; Sandhoff disease;Schilder's disease; schizencephaly; septo-optic dysplasia; shaken babysyndrome; shingles; Shy-Drager syndrome; Sjögren's syndrome; sleepapnea; Soto's syndrome; spasticity; spina bifida; spinal cord injury;spinal cord tumors; spinal muscular atrophy; Stiff-Person syndrome;stroke; Sturge-Weber syndrome; subacute sclerosing panencephalitis;subcortical arteriosclerotic encephalopathy; Sydenham chorea; syncope;syringomyelia; tardive dyskinesia; Tay-Sachs disease; temporalarteritis; tethered spinal cord syndrome; Thomsen disease; thoracicoutlet syndrome; Tic Douloureux; Todd's paralysis; Tourette syndrome;transient ischemic attack; transmissible spongiform encephalopathies;transverse myelitis; traumatic brain injury; tremor; trigeminalneuralgia; tropical spastic paraparesis; tuberous sclerosis; vasculardementia (multi-infarct dementia); vasculitis including temporalarteritis; Von Hippel-Lindau disease; Wallenberg's syndrome;Werdnig-Hoffman disease; West syndrome; whiplash; Williams syndrome;Wildon's disease; and Zellweger syndrome.

In some embodiments, the condition, disease or disorder is an autoimmunedisease. Non-limiting examples include rheumatoid arthritis, systemiclupus erythematosus, multiple sclerosis, inflammatory bowel diseases(IBDs) comprising Crohn disease (CD) and ulcerative colitis (UC), whichare chronic inflammatory conditions with polygenic susceptibility. Incertain embodiments, the condition is an inflammatory bowel disease. Incertain embodiments, the condition is Crohn's disease, autoimmunecolitis, iatrogenic autoimmune colitis, ulcerative colitis, colitisinduced by one or more chemotherapeutic agents, colitis induced bytreatment with adoptive cell therapy, colitis associated by one or morealloimmune diseases (such as graft-vs-host disease, e.g., acute graftvs. host disease and chronic graft vs. host disease), radiationenteritis, collagenous colitis, lymphocytic colitis, microscopiccolitis, and radiation enteritis. In certain of these embodiments, thecondition is alloimmune disease (such as graft-vs-host disease, e.g.,acute graft vs. host disease and chronic graft vs. host disease), celiacdisease, irritable bowel syndrome, rheumatoid arthritis, lupus,scleroderma, psoriasis, cutaneous T-cell lymphoma, uveitis, andmucositis (e.g., oral mucositis, esophageal mucositis or intestinalmucositis).

In some embodiments, modulation of the immune system by STING providesfor the treatment of diseases, including diseases caused by foreignagents. Exemplary infections by foreign agents which may be treatedand/or prevented by the method of the present invention include aninfection by a bacterium (e.g., a Gram-positive or Gram-negativebacterium), an infection by a fungus, an infection by a parasite, and aninfection by a virus. In one embodiment of the present invention, theinfection is a bacterial infection (e.g., infection by E. coli,Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella spp.,Staphylococcus aureus, Streptococcus spp., or vancomycin-resistantenterococcus). In another embodiment, the infection is a fungalinfection (e.g. infection by a mould, a yeast, or a higher fungus). Instill another embodiment, the infection is a parasitic infection (e.g.,infection by a single-celled or multicellular parasite, includingGiardia duodenalis, Cryptosporidium parvum, Cyclospora cayetanensis, andToxoplasma gondiz). In yet another embodiment, the infection is a viralinfection (e.g., infection by a virus associated with AIDS, avian flu,chickenpox, cold sores, common cold, gastroenteritis, glandular fever,influenza, measles, mumps, pharyngitis, pneumonia, rubella, SARS, andlower or upper respiratory tract infection (e.g., respiratory syncytialvirus)).

In some embodiments, the condition, disease or disorder is hepatits B(see, e.g., WO 2015/061294).

In some embodiments, the condition, disease or disorder is mucositis,also known as stomatitits, which can occur as a result of chemotherapyor radiation therapy, either alone or in combination as well as damagecaused by exposure to radiation outside of the context of radiationtherapy.

In some embodiments, the condition, disease or disorder is uveitis,which is inflammation of the uvea (e.g., anterior uveitis, e.g.,iridocyclitis or iritis; intermediate uveitis (also known as parsplanitis); posterior uveitis; or chorioretinitis, e.g., pan-uveitis).

Combination Therapy

This disclosure contemplates both monotherapy regimens as well ascombination therapy regimens.

In some embodiments, the methods described herein can further includeadministering one or more additional therapies (e.g., one or moreadditional therapeutic agents and/or one or more therapeutic regimens)in combination with administration of the compounds described herein.

In certain embodiments, the methods described herein can further includeadministering one or more additional cancer therapies.

The one or more additional cancer therapies can include, withoutlimitation, surgery, radiotherapy, chemotherapy, toxin therapy,immunotherapy, cryotherapy, cancer vaccines (e.g., HPV vaccine,hepatitis B vaccine, Oncophage, Provenge) and gene therapy, as well ascombinations thereof. Immunotherapy, including, without limitation,adoptive cell therapy, the derivation of stem cells and/or dendriticcells, blood transfusions, lavages, and/or other treatments, including,without limitation, freezing a tumor.

In some embodiments, the one or more additional cancer therapies ischemotherapy, which can include administering one or more additionalchemotherapeutic agents.

In certain embodiments, the additional chemotherapeutic agent is animmunomodulatory moiety, e.g., an immune checkpoint inhibitor. Incertain of these embodiments, the immune checkpoint inhibitor targets animmune checkpoint receptor selected from the group consisting of CTLA-4,PD-1, PD-L1, PD-1-PD-L1, PD-1-PD-L2, interleukin-2 (IL-2), indoleamine2,3-dioxygenase (IDO), IL-10, transforming growth factor-β (TGFβ), Tcell immunoglobulin and mucin 3 (TIM3 or HAVCR2), Galectin 9-TIM3,Phosphatidylserine-TIM3, lymphocyte activation gene 3 protein (LAG3),MHC class II-LAG3, 4-1BB-4-1BB ligand, OX40-OX40 ligand, GITR, GITRligand-GITR, CD27, CD70-CD27, TNFRSF25, TNFRSF25-TL1A, CD40L, CD40-CD40ligand, HVEM-LIGHT-LTA, HVEM, HVEM-BTLA, HVEM-CD160, HVEM-LIGHT,HVEM-BTLA-CD160, CD80, CD80-PDL-1, PDL2-CD80, CD244, CD48-CD244, CD244,ICOS, ICOS-ICOS ligand, B7-H3, B7-H4, VISTA, TMIGD2, HHLA2-TMIGD2,Butyrophilins, including BTNL2, Siglec family, TIGIT and PVR familymembers, KIRs, ILTs and LIRs, NKG2D and NKG2A, MICA and MICB, CD244,CD28, CD86-CD28, CD86-CTLA, CD80-CD28, CD39, CD73 Adenosine-CD39-CD73,CXCR4-CXCL12, Phosphatidylserine, TIM3, Phosphatidylserine-TIM3,SIRPA-CD47, VEGF, Neuropilin, CD160, CD30, and CD155; e.g., CTLA-4 orPD1 or PD-L1). See, e.g., Postow, M. J. Clin. Oncol. 2015, 33, 1.

In certain of these embodiments, the immune checkpoint inhibitor isselected from the group consisting of: Urelumab, PF-05082566, MEDI6469,TRX518, Varlilumab, CP-870893, Pembrolizumab (PD1), Nivolumab (PD1),Atezolizumab (formerly MPDL3280A) (PDL1), MEDI4736 (PD-L1), Avelumab(PD-L1), PDR001 (PD1), BMS-986016, MGA271, Lirilumab, IPH2201,Emactuzumab, INCB024360, Galunisertib, Ulocuplumab, BKT140, Bavituximab,CC-90002, Bevacizumab, and MNRP1685A, and MGA271.

In certain embodiments, the additional chemotherapeutic agent is a STINGagonist, e.g., a STING agonist other than a compound of formula (I) asdescribed herein. For example, the STING agonist can comprise aflavonoid. Suitable flavonoids include, but are not limited to,10-(carboxymethyl)-9(10 OH)acridone (CMA),5,6-Dimethylxanthenone-4-acetic acid (DMXAA), methoxyvone, 6,4′-dimethoxyflavone, 4′-methoxyflavone, 3′, 6′-dihydroxyflavone,7,2′-dihydroxyflavone, daidzein, formononetin, retusin 7-methyl ether,xanthone, or any combination thereof. In some aspects, the STING agonistcan be 10-(carboxymethyl)-9(10OH)acridone (CMA). In some aspects, theSTING agonist can be 5,6-Dimethylxanthenone-4-acetic acid (DMXAA). Insome aspects, the STING agonist can be methoxyvone. In some aspects, theSTING agonist can be 6, 4′-dimethoxyflavone. In some aspects, the STINGagonist can be 4′-methoxyflavone. In some aspects, the STING agonist canbe 3′, 6′-dihydroxyflavone. In some aspects, the STING agonist can be 7,2′-dihydroxyflavone. In some aspects, the STING agonist can be daidzein.In some aspects, the STING agonist can be formononetin. In some aspects,the STING agonist can be retusin 7-methyl ether. In some aspects, theSTING agonist can be xanthone. In some aspects, the STING agonist can beany combination of the above flavonoids. Thus, for example, in someembodiments the flavonoid comprises DMXAA.

In certain embodiments, the additional chemotherapeutic agent is analkylating agent. Alkylating agents are so named because of theirability to alkylate many nucleophilic functional groups under conditionspresent in cells, including, but not limited to cancer cells. In afurther embodiment, an alkylating agent includes, but is not limited to,Cisplatin, carboplatin, mechlorethamine, cyclophosphamide, chlorambucil,ifosfamide and/or oxaliplatin. In an embodiment, alkylating agents canfunction by impairing cell function by forming covalent bonds with theamino, carboxyl, sulfhydryl, and phosphate groups in biologicallyimportant molecules or they can work by modifying a cell's DNA. In afurther embodiment an alkylating agent is a synthetic, semisynthetic orderivative.

In certain embodiments, the additional chemotherapeutic agent is ananti-metabolite. Anti-metabolites masquerade as purines or pyrimidines,the building-blocks of DNA and in general, prevent these substances frombecoming incorporated in to DNA during the “S” phase (of the cellcycle), stopping normal development and division. Anti-metabolites canalso affect RNA synthesis. In an embodiment, an antimetabolite includes,but is not limited to azathioprine and/or mercaptopurine. In a furtherembodiment an anti-metabolite is a synthetic, semisynthetic orderivative.

In certain embodiments, the additional chemotherapeutic agent is a plantalkaloid and/or terpenoid. These alkaloids are derived from plants andblock cell division by, in general, preventing microtubule function. Inan embodiment, a plant alkaloid and/or terpenoid is a vinca alkaloid, apodophyllotoxin and/or a taxane. Vinca alkaloids, in general, bind tospecific sites on tubulin, inhibiting the assembly of tubulin intomicrotubules, generally during the M phase of the cell cycle. In anembodiment, a vinca alkaloid is derived, without limitation, from theMadagascar periwinkle, Catharanthus roseus (formerly known as Vincarosea). In an embodiment, a vinca alkaloid includes, without limitation,Vincristine, Vinblastine, Vinorelbine and/or Vindesine. In anembodiment, a taxane includes, but is not limited, to Taxol, Paclitaxeland/or Docetaxel. In a further embodiment a plant alkaloid or terpernoidis a synthetic, semisynthetic or derivative. In a further embodiment, apodophyllotoxin is, without limitation, an etoposide and/or teniposide.In an embodiment, a taxane is, without limitation, docetaxel and/orortataxel. [021] In an embodiment, a cancer therapeutic is atopoisomerase. Topoisomerases are essential enzymes that maintain thetopology of DNA. Inhibition of type I or type II topoisomerasesinterferes with both transcription and replication of DNA by upsettingproper DNA supercoiling. In a further embodiment, a topoisomerase is,without limitation, a type I topoisomerase inhibitor or a type IItopoisomerase inhibitor. In an embodiment a type I topoisomeraseinhibitor is, without limitation, a camptothecin. In another embodiment,a camptothecin is, without limitation, exatecan, irinotecan, lurtotecan,topotecan, BNP 1350, CKD 602, DB 67 (AR67) and/or ST 1481. In anembodiment, a type II topoisomerase inhibitor is, without limitation,epipodophyllotoxin. In a further embodiment an epipodophyllotoxin is,without limitation, an amsacrine, etoposid, etoposide phosphate and/orteniposide. In a further embodiment a topoisomerase is a synthetic,semisynthetic or derivative, including those found in nature such as,without limitation, epipodophyllotoxins, substances naturally occurringin the root of American Mayapple (Podophyllum peltatum).

In certain embodiments, the additional chemotherapeutic agent is astilbenoid. In a further embodiment, a stilbenoid includes, but is notlimited to, Resveratrol, Piceatannol, Pinosylvin, Pterostilbene,Alpha-Viniferin, Ampelopsin A, Ampelopsin E, Diptoindonesin C,Diptoindonesin F, Epsilon-Vinferin, Flexuosol A, Gnetin H, HemsleyanolD, Hopeaphenol, Trans-Diptoindonesin B, Astringin, Piceid andDiptoindonesin A. In a further embodiment a stilbenoid is a synthetic,semisynthetic or derivative.

In certain embodiments, the additional chemotherapeutic agent is acytotoxic antibiotic. In an embodiment, a cytotoxic antibiotic is,without limitation, an actinomycin, an anthracenedione, ananthracycline, thalidomide, dichloroacetic acid, nicotinic acid,2-deoxyglucose and/or chlofazimine. In an embodiment, an actinomycin is,without limitation, actinomycin D, bacitracin, colistin (polymyxin E)and/or polymyxin B. In another embodiment, an antracenedione is, withoutlimitation, mitoxantrone and/or pixantrone. In a further embodiment, ananthracycline is, without limitation, bleomycin, doxorubicin(Adriamycin), daunorubicin (daunomycin), epirubicin, idarubicin,mitomycin, plicamycin and/or valrubicin. In a further embodiment acytotoxic antibiotic is a synthetic, semisynthetic or derivative.

In certain embodiments, the additional chemotherapeutic agent isselected from endostatin, angiogenin, angiostatin, chemokines,angioarrestin, angiostatin (plasminogen fragment), basement-membranecollagen-derived anti-angiogenic factors (tumstatin, canstatin, orarrestin), anti-angiogenic antithrombin III, signal transductioninhibitors, cartilage-derived inhibitor (CDI), CD59 complement fragment,fibronectin fragment, gro-beta, heparinases, heparin hexasaccharidefragment, human chorionic gonadotropin (hCG), interferonalpha/beta/gamma, interferon inducible protein (IP-10), interleukin-12,kringle (plasminogen fragment), metalloproteinase inhibitors (TIMPs),2-methoxyestradiol, placental ribonuclease inhibitor, plasminogenactivator inhibitor, platelet factor-4 (PF4), prolactin 16 kD fragment,proliferin-related protein (PRP), various retinoids,tetrahydrocortisol-S, thrombospondin-1 (TSP-1), transforming growthfactor-beta (TGF-β), vasculostatin, vasostatin (calreticulin fragment)and the like.

In certain embodiments, the additional chemotherapeutic agent isselected from abiraterone acetate, altretamine, anhydrovinblastine,auristatin, bexarotene, bicalutamide, BMS 184476,2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide,bleomycin,N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly-1-Lproline-t-butylamide,cachectin, cemadotin, chlorambucil, cyclophosphamide,3′,4′-didehydro-4′-deoxy-8′-norvin-caleukoblastine, docetaxol,doxetaxel, cyclophosphamide, carboplatin, carmustine, cisplatin,cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC),dactinomycin, daunorubicin, decitabine dolastatin, doxorubicin(adriamycin), etoposide, 5-fluorouracil, finasteride, flutamide,hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine,lomustine (CCNU), MDV3100, mechlorethamine (nitrogen mustard),melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin,mitomycin, methotrexate, taxanes, nilutamide, onapristone, paclitaxel,prednimustine, procarbazine, RPR109881, stramustine phosphate,tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine,vindesine sulfate, and vinflunine.

In certain embodiments, the additional chemotherapeutic agent isplatinum, cisplatin, carboplatin, oxaliplatin, mechlorethamine,cyclophosphamide, chlorambucil, azathioprine, mercaptopurine,vincristine, vinblastine, vinorelbine, vindesine, etoposide andteniposide, paclitaxel, docetaxel, irinotecan, topotecan, amsacrine,etoposide, etoposide phosphate, teniposide, 5-fluorouracil, leucovorin,methotrexate, gemcitabine, taxane, leucovorin, mitomycin C,tegafur-uracil, idarubicin, fludarabine, mitoxantrone, ifosfamide anddoxorubicin. Additional agents include inhibitors of mTOR (mammaliantarget of rapamycin), including but not limited to rapamycin,everolimus, temsirolimus and deforolimus.

In still other embodiments, the additional chemotherapeutic agent can beselected from those delineated in U.S. Pat. No. 7,927,613, which isincorporated herein by reference in its entirety.

In certain embodiments, the second therapeutic agent or regimen isadministered to the subject prior to contacting with or administeringthe chemical entity (e.g., about one hour prior, or about 6 hours prior,or about 12 hours prior, or about 24 hours prior, or about 48 hoursprior, or about 1 week prior, or about 1 month prior).

In other embodiments, the second therapeutic agent or regimen isadministered to the subject at about the same time as contacting with oradministering the chemical entity. By way of example, the secondtherapeutic agent or regimen and the chemical entity are provided to thesubject simultaneously in the same dosage form. As another example, thesecond therapeutic agent or regimen and the chemical entity are providedto the subject concurrently in separate dosage forms.

In still other embodiments, the second therapeutic agent or regimen isadministered to the subject after contacting with or administering thechemical entity (e.g., about one hour after, or about 6 hours after, orabout 12 hours after, or about 24 hours after, or about 48 hours after,or about 1 week after, or about 1 month after).

Patient Selection

In some embodiments, the methods described herein further include thestep of identifying a subject (e.g., a patient) in need of suchtreatment (e.g., by way of biopsy, endoscopy, or other conventionalmethod known in the art). In certain embodiments, the STING protein canserve as a biomarker for certain types of cancer, e.g., colon cancer andprostate cancer. In other embodiments, identifying a subject can includeassaying the patient's tumor microenvironment for the absence of T-cellsand/or presence of exhausted T-cells, e.g., patients having one or morecold tumors. Such patients can include those that are resistant totreatment with checkpoint inhibitors. In certain embodiments, suchpatients can be treated with a chemical entity herein, e.g., to recruitT-cells into the tumor, and in some cases, further treated with one ormore checkpoint inhibitors, e.g., once the T-cells become exhausted.

In some embodiments, the chemical entities, methods, and compositionsdescribed herein can be administered to certain treatment-resistantpatient populations (e.g., patients resistant to checkpoint inhibitors;e.g., patients having one or more cold tumors, e.g., tumors lackingT-cells or exhausted T-cells).

Compound Preparation

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the formulae herein will be evident to those ofordinary skill in the art. For example, the compounds described hereincan be synthesized, e.g., using one or more of the methods describedherein and/or using methods described in, e.g., US 2015/0056224, thecontents of each of which are hereby incorporated by reference in theirentirety. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and R G M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof. The starting materials used inpreparing the compounds of the invention are known, made by knownmethods, or are commercially available. The skilled artisan will alsorecognize that conditions and reagents described herein that can beinterchanged with alternative art-recognized equivalents. For example,in many reactions, triethylamine can be interchanged with other bases,such as non-nucleophilic bases (e.g. diisopropylamine,1,8-diazabicycloundec-7-ene, 2,6-di-tert-butylpyridine, ortetrabutylphosphazene).

The skilled artisan will recognize a variety of analytical methods thatcan be used to characterize the compounds described herein, including,for example, ¹H NMR, heteronuclear NMR, mass spectrometry, liquidchromatography, and infrared spectroscopy. The foregoing list is asubset of characterization methods available to a skilled artisan and isnot intended to be limiting.

To further illustrate the foregoing, the following non-limiting,exemplary synthetic schemes are included. Variations of these exampleswithin the scope of the claims are within the purview of one skilled inthe art and are considered to fall within the scope of the invention asdescribed, and claimed herein. The reader will recognize that theskilled artisan, provided with the present disclosure, and skill in theart is able to prepare and use the invention without exhaustiveexamples.

The following abbreviations have the indicated meanings:

ACN=acetonitrile

BnNCO=(isocyanatomethyl)benzene

BSA=Amberlyst 15

BzCl=benzoyl chloride

CCl₄=carbon tetrachloride

CE=cyanoethyl

DCA=dichloroacetic acid

DCM=dichloromethane

DIAD=diisopropyl azodiformate

DIPEA=N,N-diethylisopropylamine

DMAP=4-(N,N-dimethylamino)pyridine

DMF=N,N-dimethylformamide

DMF-DMA=N,N-dimethylformamide dimethyl acetal

DMTrCl=1-[chloro(4-methoxyphenyl)benzyl]-4-methoxybenzene

H₂O=water

HF=hydrogen fluoride

H₂S=hydrogen sulfide

I₂=iodine

MeNH₂=methylamine

NaN₃=sodium azide

NMP=N-methylpyrrolidinone

Py or pyr=pyridine

Py.TFA=pyridinium trifluoroacetate

TBDPS=tert-butyldiphenylsilyl

TBDPSCi=tert-butyl(chloro)diphenylsilane

TEA=triethylamine

TEA.HF or TEA.3HF=triethylamine trihydrofluoride

TFA=trifluoroacetic acid

Tr or Trt=trityl

TrCl=trityl chloride or triphenylmethyl chloride

TMSCl=chlorotrimethylsilane

Synthesis of Compounds of Formula I Including Amino Linkage at 3′ RibosePositions

Scheme 1 depicts an example synthesis of cyclic dinucleotidephosphoramidates as disclosed herein that include an amino linkagebonded to the 3′ position of each ribose moiety.

The sequence initiates with the treatment of compound 1 with tritylchloride in the presence of triethylamine to produce tritylated amine 2.Amine 2 is subjected to3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine, andtrifluoroacetic acid to furnish phosphoramidite 3. Subsequent water orhydrogen sulfide treatment results in phosphonate 4a or phosphonothioate4b, respectively. Compound 1 can then be combined with either ofcompounds 4a or 4b with triethylamine in carbon tetrachloride togenerate either phosphoramidate 5a or phosphoramidothioate 5b,respectively. Subjection of compound 5a or compound 5b to3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine, andtrifluoroacetic acid produces the corresponding phosphoramidite 6a or6b. Sequential treatment of phosphoramidite 6a with water anddichloroacetic acid yields phosphonate 7a. Sequential treatment ofphosphoramidite 6b with hydrogen sulfide and dichloroacetic acid yieldsphosphonothioate 7b. Compound 7a or 7b are then taken up intriethylamine and carbon tetrachloride to enable cyclization to producecyclic phosphoramidates 8a and 8b. Lastly, treatment of compound 8a or8b with methylamine and triethylamine-hydrogen fluoride complex, inturn, result in decyanoethylated cyclic dinucleotide phosphoramidates 9aor 9b, in which the adenine and/or guanine bases have also beendeprotected.

Synthesis of Compounds of Formula I Including Amino Linkage at 5′ RibosePositions and Fluoro at 2′ Positions

Schemes 2 and 3 depict an exemplary synthesis of cyclic dinucleotidephosphoramidates as disclosed herein that include an amino linkagebonded to the 5′ position of each ribose moiety, in addition to a fluoroat the deoxy-2′ positions.

Compound 1 is treated with chlorotrimethylsilane, followed by eitherbenzoyl chloride (if B=adenine) or isobutyryl chloride (if B=guanine) toproduce compound 2. Compound 2 is combined with triphenylphosphine,iodine, and imidazole in N-methylpyrrolidinone to produce5′-hydroxylated intermediate 3. Intermediate 3 is subjected to sodiumazide in N,N-dimethylformamide to give azide 4. Azide 4 is subjected totriphenylphosphine in what is understood to be a Staudinger reaction togenerate amine 5, which is subsequently tritylated with trityl chloridein the presence of triethylamine to generate compound 6. Compound 6 issubjected to treatment with((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine, andtrifluoroacetic acid to furnish phosphoramidite 7. Subsequent water orhydrogen sulfide treatment results in phosphonate 8a or phosphonothioate8b, respectively. Compound 7 can also be converted to compound 8a bymeans of pyridinium chloride treatment.

Compound 5 can then be combined with either of compounds 8a or 8b withtriethylamine in carbon tetrachloride to generate either phosphoramidate9a or phosphoramidothioate 9b, respectively. Subjection of compound 9aor compound 9b to3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine, andtrifluoroacetic acid produces the corresponding phosphoramidite 10a or10b. Sequential treatment of phosphoramidite 10a with water anddichloroacetic acid yields phosphonate 11a. Sequential treatment ofphosphoramidite 10b with hydrogen sulfide and dichloroacetic acid yieldsphosphonothioate 11b. Compound 11a or 11b can be taken up intriethylamine and carbon tetrachloride to enable intramolecularcyclization to produce cyclic phosphoramidates 12a and 12b. Lastly,treatment of compound 12a or 12b with methylamine results indecyanoethylated cyclic dinucleotide phosphoramidates 13a or 13b, inwhich the adenine and/or guanine bases have also been deprotected.

Synthesis of Compounds of Formula I Including Amino Linkage at 5′ RibosePositions

Scheme 4 depicts an example synthesis of cyclic dinucleotidephosphoramidates as disclosed herein that include an amino linkagebonded to the 5′ position of each ribose moiety.

The sequence initiates with the treatment of compound 1 with tritylchloride in the presence of triethylamine to produce tritylated amine 2.Amine 2 is subjected to3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine, andtrifluoroacetic acid to furnish phosphoramidite 3. Subsequent water orhydrogen sulfide treatment results in phosphonate 4a or phosphonothioate4b, respectively. Compound 1 can then be combined with either ofcompounds 4a or 4b with triethylamine in carbon tetrachloride togenerate either phosphoramidate 5a or phosphoramidothioate 5b,respectively. Subjection of compound 5a or compound 5b to3-((bis(diisopropylamino)phosphanyl)oxy)propanenitrile, pyridine, andtrifluoroacetic acid produces the corresponding phosphoramidite 6a or6b. Sequential treatment of phosphoramidite 6a with water anddichloroacetic acid yields phosphonate 7a. Phosphoramidite 6a can alsobe converted to phosphonate 7a without a separate water treatment step.Sequential treatment of phosphoramidite 6b with hydrogen sulfide anddichloroacetic acid yields phosphonothioate 7b. Compound 7a or 7b can betaken up in triethylamine and carbon tetrachloride to enableintramolecular cyclization to produce cyclic phosphoramidates 8a and 8b.Lastly, treatment of compound 8a or 8b with methylamine andtrimethylamine trihydrofluoride, in turn, result in decyanoethylatedcyclic dinucleotide phosphoramidates 9a or 9b, in which the adenineand/or guanine bases have also been deprotected.

EXAMPLES Compound Preparation Key Intermediates PreparationN-(9-((2R,3R,4R,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(TNT-C)

Step 1

(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuran-3,4-diol(101): To a suspension of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)-tetrahydrofuran-3,4-diol(500 g, 1.87 mol) in pyridine (3.5 L) were added4,4-dimethylaminopyridine (22.9 g, 0.18 mol) andtert-butyl(chloro)diphenylsilane (616 g, 2.24 mol) under nitrogenatmosphere. After stirring for 1 day at ambient temperature, thereaction suspension changed to a clear solution. After total 3 days, thereaction solution was quenched by the addition of methanol (100 mL). Themixture was concentrated under reduced pressure. The residue was addedto a mixture of chloroform (1.5 L) and diethyl ether (4 L) and vigorousstirring for 2 hours. The resulting precipitate was filtered and thefilter cake was collected and dried in the air to give crude product.The crude product was added water (3 L) and vigorous stirring for 1hour. The suspension was filtered, dried under infrared light to affordthe title compound 101 as a colorless solid (937 g, 99%): ¹H NMR (400MHz, DMSO-d₆) δ 8.53 (s, 1H), 8.38 (s, 1H), 7.68-7.57 (m, 4H), 7.51-7.31(m, 6H), 5.99 (d, J=4.5 Hz, 1H), 4.59 (t, J=4.8 Hz, 1H), 4.33 (t, J=5.0Hz, 1H), 4.08 (q, J=4.5 Hz, 1H), 3.94 (dd, J=11.4, 3.7 Hz, 1H), 3.80(dd, J=11.4, 4.8 Hz, 1H), 0.98 (s, 9H); LC/MS: [(M+1)]⁺=506.2.

Step 2

(2R,3S,4S,5R)-2-(6-amino-9H-purin-9-yl)-4-bromo-5-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuran-3-ylacetate (102)

To a suspension of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuran-3,4-diol(101, 900 g, 1.78 mol) and H₂O (29.3 mL, 1.63 mol) in acetonitrile (13.5L) was added dropwise a solution of 1-bromo-2-methyl-1-oxopropan-2-ylacetate (787 mL, 5.34 mol) in acetonitrile (4.5 L) over 2 hours undernitrogen atmosphere at 0° C. Upon complete addition, the suspensionchanged to a clear solution. After total 5.5 hours, the pH value of thereaction mixture was adjusted to 6 with sodium bicarbonate. Theresulting mixture was concentrated under reduced pressure and theresidue was triturated with dichloromethane (2 L), filtered and washedwith water (1 L), dried under infrared light to give the title compound102 as a white solid (597 g, 59%): ¹H NMR (400 MHz, DMSO-d₆) δ 8.38 (s,1H), 8.28 (s, 1H), 7.73-7.63 (m, 4H), 7.55-7.36 (m, 6H), 6.24 (d, J=3.2Hz, 1H), 5.91 (t, J=3.2 Hz, 1H), 4.94 (dd, J=5.0, 3.1 Hz, 1H), 4.57 (q,J=4.9 Hz, 1H), 4.06-3.95 (m, 2H), 2.13 (s, 3H), 1.02 (s, 9H); LC/MS:[(M+1)]⁺=610.2, 612.2.

Step 3

(2R,3S,4R,5R)-2-(6-amino-9H-purin-9-yl)-4-bromo-5-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuran-3-ol(103)

To a suspension of(2R,3S,4S,5R)-2-(6-amino-9H-purin-9-yl)-4-bromo-5-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuran-3-ylacetate (102, 490 g, 0.80 mol) in 1,4-dioxane (7 L) was added butylamine(220 g, 2.06 mol). The mixture was warmed to 100° C. and stirred for 3hours, over which time the suspension changed to a clear solution. Theresulting mixture was concentrated under reduced pressure and theresidue was added to a mixture of petroleum, dichloromethane andmethanol (3.1 L, 25/5/1, v/v/v) and stirred vigorously for 1 h. Thesuspension was filtered and the filter cake was washed with water (4 L)and dried under infrared light to afford the title compound 103 as awhite solid (360 g, 79%): ¹H NMR (300 MHz, DMSO-d₆) δ 8.14 (s, 1H), 8.10(s, 1H), 7.72-7.61 (m, 4H), 7.53-7.36 (m, 6H), 7.32 (s, 2H), 6.49 (d,J=5.2 Hz, 1H), 5.91 (d, J=3.8 Hz, 1H), 4.95 (q, J=4.3 Hz, 1H), 4.61 (dd,J=5.4, 4.0 Hz, 1H), 4.54 (q, J=4.9 Hz, 1H), 4.08-3.94 (m, 2H), 1.02 (s,9H); LC/MS: [(M+1)]⁺=568.1, 570.1.

Step 4

(2R,3S,4S,5R)-2-(6-amino-9H-purin-9-yl)-4-bromo-5-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuran-3-ylbenzylcarbamate (104)

To a suspension of(2R,3S,4R,5R)-2-(6-amino-9H-purin-9-yl)-4-bromo-5-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuran-3-ol(103, 290 g, 0.51 mol) in a cosolvent of tetrahydrofuran andacetonitrile (5.8 L, 1/1, v/v) was added triethylamine (106 mL, 0.77mol) and (isocyanatomethyl)benzene (102.7 g, 0.77 mol). The resultingsuspension was stirred for 15 hours at 35° C. The reaction mixture wasquenched by the addition of methanol (300 mL). The mixture wasconcentrated under reduced pressure and the residue was triturated by amixture of petroleum ether, ethyl acetate and dichloromethane (2.2 L,5/1/1.5, v/v/v). The suspension was filtered and the filter cake wascollected, dried under infrared light to afford the title compound 104as a white solid (348 g, 97%): ¹H NMR (300 MHz, DMSO-d₆) δ 8.17-8.12 (m,3H), 8.15 (s, 1H), 8.12 (s, 1H), 7.73-7.61 (m, 4H), 7.54-7.10 (m, 13H),6.16 (d, J=4.0 Hz, 1H), 5.88 (t, J=4.1 Hz, 1H), 4.90 (dd, J=5.4, 4.2 Hz,1H), 4.53 (q, J=4.8 Hz, 1H), 4.30-4.09 (m, 2H), 4.08-3.92 (m, 2H), 1.03(s, 9H); LC/MS: [(M+1)]⁺=701.2, 703.2.

Step 5

(3aR,4S,6R,6aR)-6-(6-amino-9H-purin-9-yl)-3-benzyl-4-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuro[3,4-d]oxazol-2(3H)-one(105)

A solution of(2R,3S,4S,5R)-2-(6-amino-9H-purin-9-yl)-4-bromo-5-((tert-butyldiphenylsilyloxy)methyl)-tetrahydrofuran-3-ylbenzylcarbamate (104, 348 g, 0.50 mol) in tetrahydrofuran (10.5 L) wastreated with sodium tert-butoxide (57.2 g, 0.60 mol) for 0.5 h at −20°C. The reaction was then quenched by the addition of saturated aqueousammonium chloride (4 L). The organic phase was separated and the aqueousphase was extracted with ethyl acetate (2 L). The combined organiclayers were dried over anhydrous sodium sulfate. After filtration, thefiltrate was concentrated under reduced pressure to afford the titlecompound 105 which was used in the next step without furtherpurification (315 g, white foam): LC/MS: [(M+1)]⁺=621.2.

Step 6

N-(9-((3aR,4S,6R,6aR)-3-benzyl-4-((tert-butyldiphenylsilyloxy)methyl)-2-oxo-hexahydrofuro[3,4-d]oxazol-6-yl)-9H-purin-6-yl)isobutyramide(106)

To the solution of To the above crude compound (105, 280 g) in distilledpyridine (2.8 L) was added isobutyryl chloride (71.7 g, 0.68 mol) at 0°C. Then the mixture was warmed to room temperature and stirred for 1 h,over which time the color of the reaction mixture changed to orange. Thereaction mixture was quenched with methanol (250 mL) and concentratedunder reduced pressure to afford the crude title compound 106 as ayellow oil (311 g): LC/MS: [(M+1)]⁺=691.3.

Step 7

N-(9-((3aR,4S,6R,6aR)-3-benzyl-4-(hydroxymethyl)-2-oxo-hexahydrofuro[3,4-d]oxazol-6-yl)-9H-purin-6-yl)isobutyramide(107)

To a suspension of the above crude compound (106, 354 g) intetrahydrofuran (3 L) was added triethylamine trihydrofluoride (590 g,3.55 mol) and stirred for 17 hours at ambient temperature. Uponcompletion, the reaction mixture changed to a clear solution, which wasquenched with saturated aqueous sodium bicarbonate (2 L). The organiclayer was separated and the aqueous layer was extracted withdichloromethane (2×1 L). The organic layers were combined and dried overanhydrous sodium sulfate and filtered. The filtrate was concentratedunder reduced pressure and the residue was triturated with petroleumether and dichloromethane (2.5 L, 2:1, v/v). The resulting precipitatewas filtered and dried under infrared light to afford the title compound107 as a white solid. (124 g, 55% over 3 steps): ¹H NMR (300 MHz,DMSO-d₆) δ 10.70 (s, 1H), 8.66 (s, 1H), 8.64 (s, 1H), 7.48-7.28 (m, 5H),6.44 (d, J=3.2 Hz, 1H), 5.77 (dd, J=8.4, 3.3 Hz, 1H), 5.24-5.14 (m, 1H),4.65 (d, J=15.4 Hz, 1H), 4.46-4.27 (m, 3H), 3.44 (t, J=5.3 Hz, 2H), 2.94(h, J=6.9 Hz, 1H), 1.13 (d, J=6.8 Hz, 6H); LC/MS: [(M+1)]⁺=453.2.

Step 8

N-(9-((3aR,4S,6R,6aR)-3-benzyl-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-oxo-hexahydrofuro[3,4-d]oxazol-6-yl)-9H-purin-6-yl)isobutyramide(108)

To a solution of N-(9-((3aR,4S,6R,6aR)-3-benzyl-4-(hydroxymethyl)-2-oxo-hexahydrofuro[3,4-d]oxazol-6-yl)-9H-purin-6-yl)isobutyramide(107, 94 g, 0.21 mol) in distilled pyridine (1 L) was added1-[chloro(4-methoxyphenyl)benzyl]-4-methoxybenzene (98 g, 0.29 mol). Theresulting solution was stirred for 7 h at ambient temperature. Uponcompletion, the reaction was quenched with methanol (50 mL). Thereaction mixture was concentrated to afford the crude title compound 108as an orange oil, which was used in the next step without furtherpurification (157 g, crude oil): LC/MS: [(M+1)]⁺=755.3.

Step 9

(2R,3R,4S,5S)-2-(6-amino-9H-purin-9-yl)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methoxy)methyl)-tetrahydrofuran-3-ol(109)

A solution of the above crude product (108, 157 g) in ethanol (2 L) wastreated with 10 N aqueous solution of sodium hydroxide (450 mL) atreflux for 1 h. After cooling down to ambient temperature, the resultingsolution was concentrated to about one third volume then the pH value ofthe suspension was adjusted to 8 with saturated aqueous solution ofammonium chloride. The resulting mixture was extracted withdichloromethane (3×2 L). The organic layers were combined and dried overanhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure to afford crude title compound 109as a yellow solid, which was used in the next step reaction withoutfurther purification (137 g, crude yellow solid): LC/MS: [(M+1)]⁺=659.2.

Step 10

9-((2R,3R,4R,5S)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-tetrahydrofuran-2-yl)-9H-purin-6-amine(110)

To a solution of the above crude product (109, 137 g) in distilledpyridine (1.5 L) were added imidazole (71 g, 1.04 mol) andtert-butylchlorodimethylsilane (94 g, 0.62 mol). The resulting solutionwas stirred for 15 h at ambient temperature. The mixture wasconcentrated under reduced pressure and the residue was purified bysilica gel column chromatography, eluting with 20%˜40% petroleum etherin ethyl acetate (plus 0.1% TEA, v/v) to afford the title compound 110as an orange oil (115 g, 71%): LC/MS: [(M+1)]⁺=773.3.

Step 11

9-((2R,3R,4R,5S)-4-((Z)-benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-tetrahydrofuran-2-yl)-9H-purin-6-amine(111)

A solution of(E)-N′-(9-((2R,3R,4R,5S)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(110, 115 g, 148.8 mmol) in acetonitrile (1.5 L) was treated withdiisopropyl azodiformate (300 g, 1.5 mol) for 28 hours at 30° C. Theresulting mixture was concentrated under reduced pressure to affordcompound 111, which was used in the next step without furtherpurification (115 g, brown oil): LC/MS: [(M+1)]⁺=771.3.

Step 12

N-(9-((2R,3R,4R,5S)-4-((Z)-benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(112)

To a solution of the above crude product (111, 115 g) in distilledpyridine (1 L) was added benzoyl chloride (63 g) over 30 min at 0° C.The reaction mixture was then warmed to room temperature and stirred for3 h. The reaction mixture was quenched by the addition of methanol (50mL) and the resulting solution was concentrated under reduce pressure.The residue was dissolved in tetrahydrofuran (1 L), cooled to 0° C.,followed by the addition of ammonium hydroxide (300 mL, 27% in water).The resulting precipitate was removed by filtration. The filtrate wasconcentrated under reduce pressure to afford crude title compound 112,which was used in the next step reaction without further purification:LC/MS: [(M+1)]⁺=875.3.

Step 13

N-(9-((2R,3R,4R,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(INT-C)

The above crude solution ofN-(9-((2R,3R,4R,5S)-4-((Z)-benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-(tert-butyldimethylsilyloxy)-tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide (112, 115 g) indichloromethane (1.2 L) and methanol (120 mL) were added Amberlyst-15(300 g) and water (25 mL). After 2 hours at ambient temperature, theresulting mixture was filtered and washed with dichloromethane (2×200mL). The solids were collected and washed with a mixture ofdichloromethane, triethylamine and methanol (3×2 L, 7/2/1, v/v/v). Theorganic layers were combined and concentrated under reduced pressure andthe residue was purified by silica gel column chromatography, elutingwith 1%˜3% methanol in dichloromethane (plus 0.1% TEA, v/v) to affordthe title compound as a white solid (INT-C, 26 g, 36% over 3 steps). ¹HNMR (400 MHz, DMSO-d₆) δ 8.81 (s, 1H), 8.76 (s, 1H), 8.10-8.02 (m, 2H),7.70-7.51 (m, 3H), 6.12 (d, J=2.8 Hz, 1H), 5.77 (s, 1H), 5.21 (s, 1H),4.60 (dd, J=5.1, 2.8 Hz, 1H), 3.88 (dt, J=6.8, 3.2 Hz, 1H), 3.81 (d,J=12.0 Hz, 1H), 3.71-3.58 (m, 2H), 3.00 (q, J=7.3 Hz, 10H), 1.18 (t,J=7.3 Hz, 15H), 0.86 (s, 9H), 0.07 (s, 3H), 0.02 (s, 3H); LC/MS:[(M+1)]⁺=485.2.

N-(9-((2R,3R,4R,5S)-4-amino-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(INT-D)

Step 1

(E)-N′-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(113)

To a suspension of2-amino-9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one(360.0 g, 1.27 mol) in methanol (5 L) was added N,N-dimethylformamidedimethyl acetal (393.6 g, 3.31 mol). The resulting mixture was stirredfor 3 days at ambient temperature. Upon completion, the solids werecollected by filtration, washed with cold methanol (3×500 mL) and driedin a vacuum oven to afford the title compound as a colorless solid (113,330 g, 77%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.30 (s, 1H), 8.50 (s, 1H),8.00 (s, 1H), 5.76 (d, J=6.1 Hz, 1H), 5.37 (d, J=6.2 Hz, 1H), 5.13 (d,J=4.6 Hz, 1H), 4.98 (t, J=5.5 Hz, 1H), 4.45 (q, J=5.9 Hz, 1H), 4.08 (td,J=4.9, 3.2 Hz, 1H), 3.87 (q, J=3.8 Hz, 1H), 3.67-3.43 (m, 2H), 3.12 (s,3H), 3.00 (s, 3H); LC/MS: [(M+1)]⁺=339.1.

Step 2

(E)-N′-(9-((2R,3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(114)

To a suspension of(E)-N′-(9-((2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(113, 330 g, 0.97 mol) in pyridine (4 L) was addedtert-butyl(chloro)diphenylsilane (294 g, 1.07 mol). The resultingsolution was stirred for 3 days at ambient temperature to provide aclear solution. The solution was concentrated under reduced pressure andthe residue was triturated with water (3 L) and filtered. The filtercake was washed with water (3×1 L), diethyl ether (3×1 L) and dried in avacuum oven to afford the title compound 114 as a colorless solid (506g, 90%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.37 (s, 1H), 8.52 (s, 1H), 7.98(s, 1H), 7.74-7.56 (m, 4H), 7.53-7.29 (m, 6H), 5.86 (d, J=5.0 Hz, 1H),4.50 (t, J=5.1 Hz, 1H), 4.28 (t, J=4.9 Hz, 1H), 4.07-3.73 (m, 3H), 3.11(s, 3H), 3.03 (s, 3H), 0.99 (s, 9H); LC/MS: [(M+1)]⁺=577.2.

Step 3

(2R,3S,4S,5R)-4-bromo-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(2-((E)-((dimethylamino)methylene)amino)-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-ylacetate (115)

To a suspension of(E)-N′-(9-((2R,3R,4S,5R)-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3,4-dihydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(114, 300 g, 0.52 mol) in acetonitrile (4 L) was added water (4.7 g,0.26 mol) followed by the addition of 1-bromo-2-methyl-1-oxopropan-2-ylacetate (419 g, 2.0 mol) at 0° C. The resulting solution was stirred for4 hours at 0° C. then quenched by the addition of saturated aqueoussodium bicarbonate (3 L). The organic phase was collected and theaqueous phase was extracted with ethyl acetate (2×1 L). The organiclayers were combined and dried with anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure and theresidue was purified by silica gel column chromatography, eluting with1% methanol in dichloromethane to afford the title compound 115 as ayellow solid (269 g, 76%): ¹H NMR (400 MHz, DMSO-d₆) δ 11.37 (s, 1H),8.53 (s, 1H), 7.76 (s, 1H), 7.71-7.61 (m, 4H), 7.47-7.35 (m, 6H), 6.05(t, J=2.8 Hz, 1H), 5.99 (d, J=3.1 Hz, 1H), 4.85 (dd, J=4.7, 2.4 Hz, 1H),4.46 (q, J=5.0 Hz, 1H), 4.02-3.83 (m, 2H), 3.08 (s, 3H), 3.03-2.97 (m,3H), 2.10 (s, 3H), 0.98 (s, 9H); LC/MS: [(M+1)]⁺=681.4, 683.4.

Step 4

(E)-N′-(9-((2R,3S,4R,5R)-4-bromo-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(116)

To a solution of(2R,3S,4S,5R)-4-bromo-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(2-((E)-((dimethylamino)methylene)amino)-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-ylacetate (115, 390 g, 0.57 mol) in methanol (4 L) were addedN,N-dimethylpyridin-4-amine (3.42 g, 0.03 mol) and triethylamine (230mL, 1.71 mol). The resulting solution was stirred for 16 hours atambient temperature. Upon completion, the resulting mixture wasconcentrated under reduced pressure to afford the title compound 116 asa colorless solid which was used in the next step without furtherpurification (360 g): ¹H NMR (400 MHz, DMSO-d₆) δ 11.40 (s, 1H), 8.56(s, 1H), 7.86 (s, 1H), 7.65 (tt, J=6.2, 1.6 Hz, 4H), 7.53-7.35 (m, 6H),6.49 (d, J=5.2 Hz, 1H), 5.82 (d, J=4.0 Hz, 1H), 4.94 (q, J=4.3 Hz, 1H),4.60 (dd, J=5.4, 3.7 Hz, 1H), 4.48 (q, J=5.1 Hz, 1H), 4.04-3.91 (m, 2H),3.12 (s, 3H), 3.03 (s, 3H), 1.02 (s, 9H); LC/MS: [(M+1)]⁺=639.1, 641.1.

Step 5

(2R,3S,4S,5R)-4-bromo-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(2-((E)-((dimethylamino)methylene)amino)-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-ylbenzylcarbamate (117)

To a solution of(E)-N′-(9-((2R,3S,4R,5R)-4-bromo-5-(((tert-butyldiphenylsilyl)oxy)methyl)-3-hydroxytetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(116, 370 g, 0.58 mol) in acetonitrile (4 L) were added triethylamine(14.6 g, 0.15 mol) and (isocyanatomethyl)benzene (92.6 g, 0.70 mol). Theresulting solution was stirred for 4 hours at ambient temperature. Uponcompletion, the resulting mixture was concentrated under reducedpressure and the residue was purified by silica gel columnchromatography, eluting with 1% methanol in dichloromethane to affordthe title compound 117 as a colorless solid (317 g, 71%): ¹H NMR (300MHz, DMSO-d₆) δ 11.41 (s, 1H), 8.57 (s, 1H), 8.17 (s, 1H), 7.87-7.83 (s,1H), 7.81-7.62 (m, 4H), 7.60-7.41 (m, 6H), 7.21-7.14 (m, 4H), 6.13 (s,1H), 6.01 (s, 1H), 4.86 (dd, J=5.0, 3.1 Hz, 1H), 4.52 (q, J=5.0 Hz, 1H),4.34-4.17 (m, 1H), 3.98-3.91 (m, 1H), 3.33 (s, 2H), 3.18 (d, J=4.9 Hz,1H), 3.03 (d, J=4.7 Hz, 6H), 1.03 (s, 9H); LC/MS: [(M+1)]⁺=772.1, 774.1.

Step 6

(E)-N′-(9-((3aR,4S,6R,6aR)-3-benzyl-4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-oxohexahydrofuro[3,4-d]oxazol-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(118)

A solution of(2R,3S,4S,5R)-4-bromo-5-(((tert-butyldiphenylsilyl)oxy)methyl)-2-(2-((E)-((dimethylamino)methylene)amino)-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-ylbenzylcarbamate (117, 214 g, 0.27 mol) in tetrahydrofuran (2.5 L) wastreated with sodium tert-butoxide (79.84 g, 0.83 mol) for 1 hour atambient temperature. The reaction was then quenched by the addition ofsaturated aqueous solution of ammonium chloride (5 L). The organic layerwas separated and the aqueous layer was extracted with ethyl acetate(3×1 L). The organic layers were combined and dried over anhydroussodium sulfate. After filtration, the filtrate was concentrated underreduced pressure and the residue was purified by silica gel columnchromatography, eluting with 1% methanol in dichloromethane to affordthe title compound 118 as a colorless solid (147 g, 77%): ¹H NMR (400MHz, DMSO-d₆) δ 11.44 (s, 1H), 8.51 (s, 1H), 7.95 (s, 1H), 7.49-7.27 (m,15H), 6.31 (d, J=2.6 Hz, 1H), 6.03 (dd, J=8.4, 2.7 Hz, 1H), 4.69-4.53(m, 2H), 4.46-4.35 (m, 2H), 3.56-3.43 (m, 2H), 2.98 (d, J=7.8 Hz, 6H),0.83 (s, 9H); LC/MS: [(M+1)]⁺=692.2.

Step 7

(E)-N′-(9-((3aR,4S,6R,6aR)-3-benzyl-4-(hydroxymethyl)-2-oxohexahydrofuro[3,4-d]oxazol-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(119)

A solution of (E)-N′-(9-((3aR,4S,6R,6aR)-3-benzyl-4-(((tert-butyldiphenylsilyl)oxy)methyl)-2-oxohexahydrofuro[3,4-d]oxazol-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(118, 146 g, 0.91 mol) in tetrahydrofuran (3 L) was treated withtriethylamine trihydrofluoride (500 g, 3.5 mol) for 16 hours at ambienttemperature. The reaction was quenched with a saturated aqueous solutionof sodium bicarbonate (1 L). The organic layer was separated and theaqueous layer was extracted with ethyl acetate (2×1 L). The organiclayers were combined and dried over anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure and theresidue was purified by silica gel column chromatography, eluting with1% methanol in dichloromethane to afford the title compound 119 as acolorless solid (119 g, 87%): ¹H NMR (400 MHz, DMSO-d₆) δ 11.36 (s, 1H),8.52 (s, 1H), 8.02 (s, 1H), 7.45-7.29 (m, 5H), 6.23 (d, J=3.2 Hz, 1H),5.73 (dd, J=8.5, 3.2 Hz, 1H), 5.19-5.12 (m, 1H), 4.67 (d, J=15.4 Hz,1H), 4.46-4.20 (m, 3H), 3.47-3.37 (m, 2H), 3.03 (d, J=7.7 Hz, 6H);LC/MS: [(M+1)]⁺=454.1.

Step 8

(E)-N′-(9-((3aR,4S,6R,6aR)-3-benzyl-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-oxohexahydrofuro[3,4-d]oxazol-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(120)

To a solution of (E)-N′-(9-((3aR,4S,6R,6aR)-3-benzyl-4-(hydroxymethyl)-2-oxohexahydrofuro[3,4-d]oxazol-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(119, 55 g, 0.12 mol) in dichloromethane (500 mL) were addedtriethylamine (18 g, 0.18 mol) and1-[chloro(4-methoxyphenyl)benzyl]-4-methoxybenzene (49 g, 0.14 mol). Theresulting solution was stirred for 16 hours at ambient temperature. Uponcompletion, the reaction was quenched with a saturated aqueous solutionof sodium bicarbonate (1 L). The organic layer was separated and theaqueous layer was extracted with dichloromethane (2×200 mL). The organiclayers were combined and dried over anhydrous sodium sulfate. Afterfiltration, the filtrate was concentrated under reduced pressure and theresidue was purified by silica gel column chromatography, eluting with1% methanol in dichloromethane (plus 0.1% TEA, v/v) to afford the titlecompound 120 as a red solid (71 g, 78%): ¹H NMR (400 MHz, DMSO-d₆) δ11.37 (s, 1H), 8.30 (s, 1H), 7.95 (s, 1H), 7.32 (q, J=3.6 Hz, 3H),7.26-7.12 (m, 7H), 7.10-6.98 (m, 4H), 6.79-6.68 (m, 4H), 6.32 (d, J=2.4Hz, 1H), 5.93 (dd, J=8.3, 2.4 Hz, 1H), 4.66 (d, J=15.8 Hz, 1H),4.52-4.30 (m, 3H), 3.73 (d, J=2.3 Hz, 6H), 3.04 (td, J=10.2, 6.2 Hz,1H), 2.95 (s, 3H), 2.86 (s, 3H), 2.76 (dd, J=9.9, 5.5 Hz, 1H); LC/MS:[(M+1)]⁺=756.2.

Step 9

2-amino-9-((2R,3R,4S,5S)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrofuran-2-yl)-1H-purin-6(9H)-one(121)

A solution of(E)-N′-(9-((3aR,4S,6R,6aR)-3-benzyl-4-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-2-oxohexahydrofuro[3,4-d]oxazol-6-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(120, 90 g, 0.12 mol) in methanol (1 L) was treated with a 10 N aqueoussolution of sodium hydroxide (400 mL) for 16 hours at 55° C. Aftercooling down to ambient temperature, the resulting solution wasconcentrated to about one third volume then the pH value of thesuspension was adjusted to 8˜9 with 4 N hydrochloric acid (1 L). Theresulting mixture was extracted with ethyl acetate (2×500 mL). Theorganic layers were combined and dried over anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressureand the residue was purified by silica gel column chromatography,eluting with 1% to 2% methanol in dichloromethane (plus 0.1% TEA, v/v)to afford the title compound 121 as a red solid (66 g, 83%): ¹H NMR (400MHz, DMSO-d₆) δ 10.65 (s, 1H), 7.78 (s, 1H), 7.36-7.15 (m, 14H),6.88-6.78 (m, 4H), 6.48 (s, 2H), 5.90 (d, J=4.9 Hz, 1H), 5.82 (d, J=2.5Hz, 1H), 4.54 (td, J=4.8, 2.5 Hz, 1H), 3.91 (ddd, J=7.5, 4.7, 2.6 Hz,1H), 3.80 (d, J=13.5 Hz, 1H), 3.71 (s, 6H), 3.70 (d, J=11.9 Hz, 1H),3.48 (d, J=9.3 Hz, 1H), 3.24 (dd, J=10.6, 2.7 Hz, 1H), 3.16 (dd, J=10.4,4.8 Hz, 1H), 2.25 (s, 1H); LC/MS: [(M+1)]⁺=675.2.

Step 10

2-amino-9-((2R,3R,4R,5S)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one(122)

To a solution of2-amino-9-((2R,3R,4S,5S)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-hydroxytetrahydrofuran-2-yl)-1H-purin-6(9H)-one(90 g, 0.13 mol) in pyridine (1 L) were added imidazole (121, 45 g, 0.67mol) and tert-butylchlorodimethylsilane (50 g, 0.33 mol). The resultingsolution was stirred for 16 hours at ambient temperature then quenchedwith methanol (10 mL). The resulting mixture was concentrated underreduced pressure and the residue was purified by silica gel columnchromatography, eluting with 1% to 2% methanol in dichloromethane (plus0.1% TEA, v/v) to afford the title compound 122 as a yellow solid (75 g,71%): ¹H NMR (400 MHz, DMSO-d₆) δ 10.50 (s, 1H), 7.82 (s, 1H), 7.45-7.11(m, 14H), 6.86 (dd, J=8.8, 2.7 Hz, 4H), 6.43 (s, 2H), 5.83 (d, J=4.2 Hz,1H), 4.75 (t, J=4.8 Hz, 1H), 3.98 (dd, J=6.1, 2.9 Hz, 1H), 3.77-3.73 (m,8H), 3.47-3.14 (m, 3H), 2.05 (q, J=6.5 Hz, 1H), 0.80 (s, 9H), 0.00 (s,3H), −0.08 (s, 3H); LC/MS: [(M+1)]⁺=789.3.

Step 11

(E)-N′-(9-((2R,3R,4R,5S)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(123)

To a solution of2-amino-9-((2R,3R,4R,5S)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one(122, 100 g, 0.13 mol) in methanol (1 L) was added N,N-dimethylformamidedimethyl acetal (52 g, 0.44 mol). The resulting solution was stirred for24 hours at ambient temperature. Upon completion, the resulting mixturewas concentrated under reduced pressure to afford the title compound 123which was used in the next step without further purification (100 g,yellow solid): LC/MS: [(M+1)]⁺=844.7.

Step 12

(1E)-N′-(9-((2R,3R,4R,5S)-4-(benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(124)

A solution of(E)-N′-(9-((2R,3R,4R,5S)-4-(benzylamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(123, 59 g, 69.90 mmol) in acetonitrile (600 mL) was treated withdiisopropyl azodiformate (141 g, 699 mmol) for 18 hours at ambienttemperature. The resulting mixture was concentrated under reducedpressure and the residue was eluted through a short silica gel column toremove the excess of diisopropyl azodiformate (eluted with 10% ethylacetate in petroleum ether plus 0.1% triethylamine, then 2% methanol indichloromethane plus 0.1% TEA) to afford the crude title compound 124which was used in the next step without further purification (89 g,brown oil): LC/MS: [(M+1)]⁺=842.4.

Step 13

2-amino-9-((2R,3R,4R,5S)-4-(benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one(125)

The above crude(1E)-N′-(9-((2R,3R,4R,5S)-4-(benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)-N,N-dimethylformimidamide(55 g) was treated with methylamine (124, 300 ml, 30% solution inethanol) for 10 min at ambient temperature. Volatiles were distilled outunder reduced pressure to give the crude title compound 125 which wasused in the next step without further purification (45 g, yellow oil):LC/MS: [(M+1)]⁺=699.3.

Step 14

N-(9-((2R,3R,4R,5S)-4-(benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(126)

The above crude2-amino-9-((2R,3R,4R,5S)-4-(benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-1H-purin-6(9H)-one(125, 45 g) was dissolved in dry pyridine (500 mL), cooled to 0˜5° C.,then subjected to the addition of isobutyryl chloride (13.5 g, 0.13mol). After 1 hour, the reaction was quenched with methanol (50 mL) andconcentrated under reduced pressure to afford the crude title compound126 as a brown oil (57 g): LC/MS: [(M+1)]⁺=787.3.

Step 15

N-(9-((2R,3R,4R,5S)-4-amino-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(INT-D)

To the above crude solution ofN-(9-((2R,3R,4R,5S)-4-(benzylideneamino)-5-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)-3-((tert-butyldimethylsilyl)oxy)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(126, 57 g) in dichloromethane (600 mL) and methanol (60 mL) were addedAmberlyst-15 (114 g) and water (14 mL). After 2 hours at ambienttemperature, the resulting mixture was filtered and washed with 10%methanol in dichloromethane (300 mL). The solids were collected andwashed with 20% trimethylamine in dichloromethane (3×500 mL). Thecombined organic layers were concentrated under reduced pressure and theresidue was purified by silica gel column chromatography, eluting with1%˜4% methanol in dichloromethane to afford the title compound INT-D asa colorless solid (10 g, 31% over 4 steps): ¹H NMR (400 MHz, DMSO-d₆) δ11.59 (s, 1H), 8.28 (s, 1H), 5.88 (d, J=4.6 Hz, 1H), 5.01 (s, 1H), 4.50(t, J=5.0 Hz, 1H), 3.78 (q, J=4.0 Hz, 1H), 3.69 (d, J=11.8 Hz, 1H),3.62-3.48 (m, 2H), 2.79 (m, 1H), 1.13 (d, J=6.8 Hz, 6H), 0.81 (s, 9H),0.01 (s, 3H), −0.12 (s, 3H); LC/MS: [(M+1)]⁺=467.2.

N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-((tritylamino)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(INT-E)

Step 1

N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(127)

To a suspension of(2R,3R,4R)-5-(6-amino-9H-purin-9-yl)-4-fluoro-2-(hydroxymethyl)oxolan-3-ol(20.0 g, 74.3 mmol) in pyridine (400 mL) was added trimethylsilylchloride (40.2 g, 372.2 mmol) at 0° C. in 5 min. The suspension wasstirred at ambient temperature for 30 min followed by the addition ofbenzoyl chloride (31.2 g, 233.1 mmol) at 0° C. in 5 min. The suspensionwas stirred at ambient temperature for 2 hours. Upon completion, thereaction was quenched with cold water (100 mL) and an aqueous solutionof ammonia (240 mL, 25% w/w). The resulting mixture was stirred for 20min at ambient temperature and was then concentrated under reducedpressure. The residue was purified by silica gel column chromatography,eluting with 10% methanol in dichloromethane to afford the titlecompound 127 as a colorless solid (25 g, 45%): ¹H NMR (300 MHz, DMSO-d₆)δ 11.25 (s, 1H), 8.78 (s, 1H), 8.72 (s, 1H), 8.12-7.99 (m, 2H),7.74-7.49 (m, 3H), 6.39 (dd, J=17.2, 2.4 Hz, 1H), 5.77 (d, J=6.2 Hz,1H), 5.53 (ddd, J=52.8, 4.4, 2.4 Hz, 1H), 5.17 (t, J=5.4 Hz, 1H),4.68-4.44 (m, 1H), 4.02 (dt, J=6.9, 3.4 Hz, 1H), 3.80 (d, J=12.4 Hz,1H), 3.62 (dt, J=12.5, 4.5 Hz, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ−203.61; LC/MS (ESI, m/z): [(M+1)]⁺=374.0.

Step 2

N-(9-((2R,3R,4R,5S)-3-fluoro-4-hydroxy-5-(iodomethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(128)

To a mixture ofN-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(127, 19.3 g, 51.6 mmol) and triphenylphosphine (33.8 g, 128.9 mmol) inpyridine (350 mL) was added a solution of iodine (28.8 g, 113.6 mmol) inpyridine (50 mL) below 20° C. over 10 min. The resulting dark solutionwas stirred for 16 hours at ambient temperature. Then it was quenchedwith saturated aqueous solution of sodium hyposulfite (800 mL) andextracted with ethyl acetate (3×500 mL). The combined organic layers waswashed with brine (800 mL) and dried over anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressure.The crude product was recrystallized from methanol to afford the puretitle compound 128 as a brown solid (15 g, 60%): ¹H NMR (300 MHz,DMSO-d₆) δ 11.27 (s, 1H), 8.79 (s, 1H), 8.67 (s, 1H), 8.12-8.00 (m, 2H),7.73-7.49 (m, 3H), 6.42 (dd, J=19.7, 2.1 Hz, 1H), 6.04 (d, J=6.3 Hz,1H), 5.74 (ddd, J=52.5, 4.7, 2.1 Hz, 1H), 4.58 (dq, J=18.7, 5.9 Hz, 1H),3.96 (td, J=7.0, 3.9 Hz, 1H), 3.68 (dd, J=11.0, 3.9 Hz, 1H), 3.50 (dd,J=11.0, 6.6 Hz, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ −201.53; LC/MS (ESI,m/z): [(M+1)]⁺=484.0.

Step 3

N-(9-((2R,3R,4R,5R)-5-(azidomethyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(129)

To a solution ofN-(9-((2R,3R,4R,5S)-3-fluoro-4-hydroxy-5-(iodomethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(128, 15 g, 31.1 mmol) in N-methyl pyrrolidone (220 mL) was added sodiumazide (4.1 g, 62.2 mmol). The resulting mixture was stirred for 8 hoursat ambient temperature. Upon completion, the resulting solution waspoured into diethyl ether (4 L). Solids were precipitated and collectedby filtration to afford the title compound 129 as a brown solid (9.2 g,74%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.25 (s, 1H), 8.80 (s, 1H), 8.67 (s,1H), 8.11-8.01 (m, 2H), 7.72-7.50 (m, 3H), 6.43 (dd, J=19.9, 2.0 Hz,1H), 5.94 (d, J=6.4 Hz, 1H), 5.68 (ddd, J=52.5, 4.8, 2.0 Hz, 1H), 4.79(dddd, J=21.2, 7.8, 6.6, 4.7 Hz, 1H), 4.14 (ddd, J=7.9, 5.4, 2.5 Hz,1H), 3.77 (dd, J=13.7, 2.9 Hz, 1H), 3.59 (dd, J=13.6, 5.7 Hz, 1H); ¹⁹FNMR (282 MHz, DMSO-d₆) δ −201.66; LC/MS (ESI, m/z): [(M+1)]⁺=399.0.

Step 4

N-(9-((2R,3R,4R,5R)-5-(aminomethyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(130)

To a solution ofN-(9-((2R,3R,4R,5R)-5-(azidomethyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(129, 16.1 g, 40.4 mmol) in tetrahydrofuran (160 mL) and water (32 mL)was added triphenylphosphine (31.7 g, 120.8 mmol). The resultingsolution was stirred for 3 hours at ambient temperature. The resultingsolution was diluted with methanol (200 mL) and filtered. The filtercake was collected to afford the title compound 130 as a colorless solid(13 g, 86%): ¹H NMR (300 MHz, DMSO-d₆) δ 8.76 (s, 1H), 8.75 (s, 1H),8.10-7.98 (m, 2H), 7.72-7.47 (m, 3H), 6.35 (dd, J=17.9, 2.7 Hz, 1H),5.59 (ddd, J=52.9, 4.7, 2.8 Hz, 1H), 4.57 (ddd, J=18.8, 7.0, 4.8 Hz,1H), 3.95 (dd, J=7.3, 3.8 Hz, 1H), 2.94 (dd, J=13.8, 3.8 Hz, 1H), 2.80(dd, J=13.8, 5.2 Hz, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ −203.45; LC/MS(ESI, m/z): [(M+1)]⁺=373.0.

Step 5

N-(9-((2R,3R,4R,5R)-3-fluoro-4-hydroxy-5-((tritylamino)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(INT-E)

To a solution ofN-(9-((2R,3R,4R,5R)-5-(aminomethyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(130, 3.0 g, 8.1 mmol) in pyridine (30 mL) was added triethylamine (1.2g, 12.1 mmol) and triphenylmethyl chloride (2.4 g, 8.6 mmol). Theresulting solution was stirred for 3 hours at ambient temperature. Thereaction was then quenched by the addition of methanol (0.2 mL) andconcentrated under reduced pressure. The residue was purified by silicagel column chromatography, eluting with 20%˜40% ethyl acetate inpetroleum ether to afford the title compound INT-E as a colorless solid(4.8 g, 98%): ¹H NMR (400 MHz, DMSO-d₆) δ 11.22 (s, 1H), 8.63 (s, 1H),8.39 (s, 1H), 8.09-7.98 (m, 2H), 7.71-7.61 (m, 1H), 7.55 (dd, J=8.3, 7.0Hz, 2H), 7.44-7.34 (m, 6H), 7.32-7.10 (m, 9H), 6.36 (dd, J=18.3, 2.9 Hz,1H), 5.85-5.60 (m, 2H), 4.87 (dq, J=17.7, 5.9 Hz, 1H), 4.19-4.09 (m,1H), 2.88 (dd, J=9.8, 6.1 Hz, 1H), 2.43 (ddd, J=15.1, 9.9, 5.4 Hz, 1H),2.32 (ddd, J=12.6, 6.1, 3.2 Hz, 1H); ¹⁹F NMR (376 MHz, DMSO-d₆) δ−203.14; LC/MS (ESI, m/z): [(M+1)]⁺=615.0.

Analog Preparation Diammonium(1S,6S,8R,9R,10S,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate)

Step 1

((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) diisopropylphosphoramidite (132)

To a solution ofN-(9-((2R,3R,4R,5S)-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-4-(tritylamino)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(131, 700 mg, 0.98 mmol) in acetonitrile (5 mL) were added3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (595 mg, 1.97mmol) and pyridinium trifluoroacetate (285 mg, 1.48 mmol) The resultingsolution was stirred for 45 min at ambient temperature. The resultingsolution of compound 132 was used in the next step without work up:LC/MS (ESI, m/z): [(M+1)]⁺=909.4.

Step 2

((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) phosphonate (133)

To the above solution of compound 132 were added water (0.18 g, 10 mmol)and pyridinium trifluoroacetate (0.28 g, 1.48 mmol). The resultingsolution was stirred for 30 min at ambient temperature. Upon completion,the resulting solution was applied onto a reversed phase C18 column,eluting with 70%˜95% (25 min) acetonitrile in water to afford the titlecompound 133 as a colorless solid (0.64 g, over two steps 78%): ¹H NMR(400 MHz, DMSO-d₆) δ 12.12 (s, 1H), 11.40 (d, J=5.9 Hz, 1H), 8.16 (d,J=12.5 Hz, 1H), 7.57-7.45 (m, 6H), 7.37 (t, J=7.6 Hz, 6H), 7.32-7.22 (m,3H), 6.18 (dd, J=6.5, 4.4 Hz, 1H), 4.60 (dt, J=19.0, 6.0 Hz, 1H),4.20-4.07 (m, 1H), 4.02 (ddt, J=11.6, 8.5, 5.8 Hz, 2H), 3.79-3.64 (m,2H), 2.95 (dt, J=4.8, 2.1 Hz, 1H), 2.89-2.73 (m, 3H), 1.15 (dd, J=6.9,3.0 Hz, 6H), 0.77 (s, 9H), −0.11 (d, J=3.3 Hz, 3H), −0.40 (d, J=9.8 Hz,3H); ³¹P NMR (162 MHz, DMSO-d₆) δ 9.92, 9.09; LC/MS (ESI, m z):[(M+1)]⁺=826.3.

Step 3

((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl)((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)phosphoramidate(134)

To a mixture of[(2S,3R,4R,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3-[(triphenylmethyl)amino]oxolan-2-yl]methyl2-cyanoethyl phosphonate (133, 640 mg, 0.71 mmol) andN-(9-((2R,3R,4R,5S)-4-amino-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(INT-C, 375 mg, 0.71 mmol) in acetonitrile (8 mL) were addedN,N-diisopropylethylamine (200 mg, 1.55 mmol) and carbon tetrachloride(477 mg, 3.10 mmol). The resulting mixture was stirred for 30 min atambient temperature. Upon completion, the resulting mixture was appliedonto a reversed phase C18 column, eluting with 70%˜95% (25 min)acetonitrile in water to afford the title compound 134 as a colorlesssolid (440 mg, 43%): ¹H NMR (400 MHz, DMSO-d₆) δ 12.07 (d, J=8.7 Hz,1H), 11.45 (d, J=10.8 Hz, 1H), 11.17 (d, J=5.0 Hz, 1H), 8.77-8.68 (m,1H), 8.17 (d, J=5.4 Hz, 1H), 8.04 (dd, J=13.6, 7.7 Hz, 2H), 7.68-7.59(m, 1H), 7.59-7.40 (m, 9H), 7.30 (td, J=8.1, 3.1 Hz, 6H), 7.20 (q, J=7.5Hz, 4H), 6.30-5.95 (m, 2H), 5.24-4.97 (m, 2H), 4.64-4.53 (m, 1H),4.52-4.37 (m, 1H), 4.07 (d, J=16.0 Hz, 1H), 4.02-3.70 (m, 5H), 3.56 (q,J=16.1, 12.7 Hz, 2H), 3.23 (s, 1H), 2.85-2.65 (m, 4H), 1.07 (dt, J=9.8,5.8 Hz, 6H), 0.91-0.59 (m, 18H), 0.02-−0.25 (m, 9H), −0.50 (d, J=26.5Hz, 3H); ³¹P NMR (162 MHz, DMSO-d₆) δ 8.72, 8.62; LC/MS (ESI, m/z):[(M+1)]⁺=1308.5.

Step 4

((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl)((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-((((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)methyl)tetrahydrofuran-3-yl)phosphoramidate(135)

To a solution of ((2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-3-(tritylamino)-tetrahydrofuran-2-yl)methyl2-cyanoethyl(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-(tert-butyldimethylsilyloxy)-2-(hydroxymethyl)-tetrahydrofuran-3-ylphosphoramidate(134, 440 mg, 0.34 mmol) in acetonitrile (2 mL) were added3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (203 mg, 0.67mmol) and pyridinium trifluoroacetate (97 mg, 0.50 mmol). The resultingmixture was stirred for 45 min at ambient temperature and was used inthe next step directly.

Step 5

((2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-3-(tritylamino)-tetrahydrofuran-2-yl)methyl2-cyanoethyl(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-(tert-butyldimethylsilyloxy)-2-(((2-cyanoethoxy)hydrophosphoryloxy)methyl)-tetrahydrofuran-3-ylphosphoramidate(136)

To the solution from the previous step were added water (0.06 g, 3.36mmol) and pyridinium trifluoroacetate (0.10 g, 0.51 mmol). The resultingsolution was stirred for 45 min at ambient temperature. Upon completion,the resulting solution was applied onto a reversed phase C18 column,eluting with 70%˜95% (25 min) acetonitrile in water to afford the titlecompound 136 as a colorless solid (400 mg, 83%): ¹H NMR (400 MHz,DMSO-d₆) δ 12.09 (d, J=9.1 Hz, 1H), 11.48-11.38 (m, 1H), 11.20 (d, J=3.2Hz, 1H), 8.79-8.51 (m, 2H), 8.24-8.14 (m, 1H), 8.08-7.99 (m, 2H),7.84-7.12 (m, 20H), 6.28-5.88 (m, 2H), 5.09 (q, J=9.7, 8.5 Hz, 1H),4.77-4.58 (m, 1H), 4.46-3.72 (m, 10H), 3.60 (t, J=11.7 Hz, 1H), 3.25 (d,J=15.0 Hz, 1H), 2.91-2.67 (m, 6H), 1.25-1.05 (m, 6H), 0.86-0.66 (m,18H), 0.01-−0.18 (m, 10H), −0.48 (d, J=24.4 Hz, 2H); ³¹P NMR (162 MHz,DMSO-d₆) δ 9.75, 9.71, 9.23, 9.14, 8.49, 8.34; LC/MS (ESI, m/z):[(M+1)]⁺=1425.5.

Step 6

((2S,3R,4R,5R)-3-amino-4-(tert-butyldimethylsilyloxy)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-tetrahydrofuran-2-yl)methyl2-cyanoethyl(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-(tert-butyldimethylsilyloxy)-2-(((2-cyanoethoxy)hydrophosphoryloxy)methyl)-tetrahydrofuran-3-ylphosphoramidate(137)

To a solution of((2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-3-(tritylamino)-tetrahydrofuran-2-yl)methyl2-cyanoethyl(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-(tert-butyldimethylsilyloxy)-2-(((2-cyanoethoxy)hydrophosphoryloxy)methyl)-tetrahydrofuran-3-ylphosphoramidate(136, 400 mg, 0.28 mmol) in dichloromethane (8 mL) were added water (25mg, 1.40 mmol) and dichloroacetic acid (640 mg, 2.81 mmol). Theresulting solution was stirred for 10 min at ambient temperature. Uponcompletion, the reaction was quenched by the addition of saturatedaqueous sodium bicarbonate (30 mL). The resulting mixture was extractedwith dichloromethane (3×30 mL). The organic layers were combined anddried over anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure to afford the title compound 137,which was used in the next step without further purification.

Step 7

N-{9-[(1R,6S,8R,9R,10R,15S,17R,18R)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-bis(2-cyanoethoxy)-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(138)

To a solution of the above crude compound (137, 332 mg) in acetonitrile(56 mL) were added triethylamine (1.1 mL) and carbon tetrachloride (1.1mL). The resulting solution was stirred for 10 min at ambienttemperature. Upon completion, the resulting solution was concentratedunder reduced pressure to afford the crude title compound 138, which wasused in the next step without further purification.

Step 8

(1R,6S,8R,9R,10R,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate);bis(methanaminium) (139)

The above crude compound 138 was treated with a solution of methyaminein ethanol (14 mL, 30%, w/w) for 30 min at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure to afford the title compound 139 as a colorless solid, whichwas used in the next step without further purification: LC/MS (ESI,m/z): [(M−2MeNH₂+1)]⁺=901.4.

Step 9

Diammonium(1S,6S,8R,9R,10S,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate)(71)

To a solution of the above crude compound 139 in pyridine (3 mL) wereadded triethylamine (1 mL) and triethylamine trihydrofluoride (2.26 g,14.00 mmol). The resulting solution was stirred for 2 hours at 60° C.After cooling down to ambient temperature, acetone (56 mL) was added toprecipitate the crude product which was purified by Prep-HPLC under thefollowing conditions: Column: XBridge Prep OBD C18 Column, 19*250 mm, 5um; Mobile phase A: Water (plus 50 mmol/L of NH₄HCO₃); Mobile phase B:ACN. Detector: 254/220 nm; Gradient: 0% B 7 min, 0%˜20% B in 30 min;Flow rate: 20 mL/min. Retention time: 17.83 min. to afford the titlecompound 71 as a colorless solid (66.9 mg, 35%): ¹H NMR (400 MHz, D₂O) δ8.43 (s, 1H), 8.17 (d, J=48.1 Hz, 2H), 5.98 (s, 1H), 5.81 (s, 1H), 4.54(s, 1H), 4.33 (s, 1H), 4.20 (t, J=14.8 Hz, 2H), 4.14-3.98 (m, 4H), 3.87(s, 2H); ³¹P NMR (162 MHz, D₂O) δ 6.58; LC/MS (ESI, m/z):[(M−2NH₃−1)]⁻=671.0.

Diammonium[(1S,6S,8R,9R,10S,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide

Step 1

N-(9-((2R,3R,4R,5S)-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-4-(tritylamino)tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-1H-purin-2-yl)isobutyramide(140)

To a solution ofN-[9-[(2R,3R,4R,5S)-4-amino-3-[(tert-butyldimethylsilyl)oxy]-5-(hydroxymethyl)oxolan-2-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl]-2-methylpropanamide(INT-D, 5.0 g, 10.72 mmol) in pyridine (96 mL) were added triethylamine(3 mL, 21.44 mmol) and (chlorodiphenylmethyl)benzene (4.6 g, 16.50mmol). The resulting solution was stirred for 16 hours at ambienttemperature, and then quenched by the addition of methanol (10 mL) andsaturated aqueous sodium bicarbonate (2 mL). The resulting mixture wasconcentrated under reduced pressure and the residue was applied onto asilica gel column, eluting with 60% ethyl acetate in petroleum ether toafford the title compound 140 as a colorless solid (5.2 g, 68%): ¹H NMR(400 MHz, DMSO-d₆) δ 12.09 (s, 1H), 11.56 (s, 1H), 8.27 (s, 1H),7.54-7.42 (m, 5H), 7.44-7.17 (m, 10H), 6.17 (d, J=6.3 Hz, 1H), 5.02 (t,J=4.7 Hz, 1H), 4.43 (t, J=5.8 Hz, 1H), 3.62 (q, J=2.5 Hz, 1H), 3.36-3.24(m, 1H), 3.23-3.13 (m, 1H), 2.98 (dt, J=4.5, 2.0 Hz, 1H), 2.84 (p, J=6.8Hz, 1H), 1.23-1.09 (m, 6H), 0.75 (s, 9H), −0.15 (s, 3H), −0.39 (s, 3H).LC/MS (ESI, m/z): [(M+1)]⁺=709.4.

Step 2

((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) diisopropylphosphoramidite (141)

To a solution ofN-[9-[(2R,3R,4R,5S)-3-[(tert-butyldimethylsilyl)oxy]-5-(hydroxymethyl)-4-[(triphenylmethyl)amino]oxolan-2-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl]-2-methylpropanamide(140, 1.50 g, 2.12 mmol) and3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (1.70 g, 5.64mmol) in acetonitrile (6 mL) was added pyridinium trifluoroacetate (0.82g, 4.25 mmol). The resulting solution was stirred for 45 min at ambienttemperature and was used in the next step directly without any workup:MS (ESI, m/z): [(M+1)]⁺=909.4.

Step 3

O-(((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl)O-(2-cyanoethyl)phosphonothioate (142)

In the above reaction solution was bubbled hydrogen sulfide for 1 minfollowed by the addition of pyridinium trifluoroacetate (820 mg, 4.25mmol). The resulting solution was stirred for 30 min at ambienttemperature and applied onto a reversed phase C18 column, eluting with70%˜95% acetonitrile (25 min) in water to afford the title compound 142as a colorless solid (1.0 g, two steps 85%): ¹H NMR (400 MHz, DMSO-d₆) δ12.12 (d, J=5.0 Hz, 1H), 11.46 (s, 1H), 8.13 (d, J=3.8 Hz, 1H),7.53-7.42 (m, 6H), 7.41-7.19 (m, 10H), 6.26-6.13 (m, 1H), 4.60 (dt,J=21.3, 6.0 Hz, 1H), 4.13-3.94 (m, 3H), 3.82-3.75 (m, 0.4H), 3.59 (t,J=6.0 Hz, 0.4H), 3.30 (d, J=2.5 Hz, 1H), 2.85 (dddd, J=30.2, 18.9, 9.2,4.7 Hz, 4H), 1.29-1.08 (m, 6H), 0.76 (d, J=3.2 Hz, 9H), −0.09-−0.19 (m,3H), −0.34-−0.51 (m, 3H); ³¹P NMR (162 MHz, DMSO) δ 72.81, 72.40; LC/MS(ESI, m/z): [(M+1)]⁺=842.3.

Step 4

O-(((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl)O-(2-cyanoethyl)((2S,3R,4R,5R)-((2S,3R,45-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)phosphoramidothioate(143)

To a mixture of[(3S,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3-[(triphenylmethyl)amino]oxolan-2-yl]methyl2-cyanoethyl sulfanylidenephosphonite (142, 0.60 g, 0.71 mmol) andN-9-[(2R,4S)-4-amino-3-[(tert-butyldimethylsilyl)oxy]-5-(hydroxymethyl)oxolan-2-yl]-9H-purin-6-ylbenzamide(INT-C, 0.35 g, 0.71 mmol) in acetonitrile (8 mL) were addedN,N-diisopropylethylamine (0.24 mL, 1.42 mmol) and carbon tetrachloride(0.27 mL, 2.84 mmol). The resulting solution was stirred for 30 min atambient temperature and applied onto a reversed phase C18 column, elutedwith 70%˜99% acetonitrile (25 min) in water to afford two isomers of thetitle compound as a colorless foam: isomer A (0.33 g, 35%, eluted outwith 95% acetonitrile): ¹H NMR (300 MHz, DMSO-d₆) δ 12.06 (s, 1H), 11.40(s, 1H), 11.16 (s, 1H), 8.68 (d, J=6.6 Hz, 2H), 8.14 (s, 1H), 8.01 (d,J=7.6 Hz, 2H), 7.69-7.15 (m, 20H), 6.18 (d, J=6.4 Hz, 1H), 6.06 (d,J=3.6 Hz, 1H), 5.49 (t, J=10.4 Hz, 1H), 5.06 (t, J=5.4 Hz, 1H), 4.71 (d,J=6.6 Hz, 1H), 4.55 (t, J=4.6 Hz, 1H), 3.74 (dt, J=28.9, 9.0 Hz, 2H),3.58 (d, J=11.7 Hz, 1H), 3.41 (d, J=14.1 Hz, 1H), 3.29 (s, 4H), 2.74 (p,J=7.4, 6.6 Hz, 4H), 1.28-1.16 (m, 1H), 1.08 (dd, J=6.8, 2.4 Hz, 6H),0.69 (d, J=11.4 Hz, 18H), −0.17 (d, J=3.0 Hz, 9H), −0.44 (s, 3H); ³¹PNMR (121 MHz, DMSO-d₆) δ 73.08; LC/MS (ESI, m/z): [(M+1)]⁺=1324.5. Andisomer B (0.35 g, 37%, eluted out with 99% acetonitrile): ¹H NMR (300MHz, DMSO-d₆) δ 12.09 (s, 1H), 11.40 (s, 1H), 11.13 (s, 1H), 8.69 (d,J=1.4 Hz, 2H), 8.10-7.96 (m, 3H), 7.67-7.38 (m, 9H), 7.22 (dt, J=33.9,7.3 Hz, 10H), 6.10 (t, J=5.3 Hz, 2H), 5.61 (t, J=10.5 Hz, 1H), 5.17 (t,J=5.2 Hz, 1H), 4.56 (t, J=4.6 Hz, 1H), 4.24 (t, J=5.5 Hz, 1H), 3.96(ddt, J=24.1, 20.9, 6.8 Hz, 6H), 3.71 (dd, J=14.9, 8.7 Hz, 2H), 3.49(dt, J=12.4, 4.4 Hz, 1H), 3.17 (d, J=2.8 Hz, 1H), 2.78 (h, J=7.9, 7.3Hz, 4H), 1.09 (dd, J=6.9, 1.5 Hz, 6H), 0.73 (d, J=16.6 Hz, 18H), −0.09(d, J=15.8 Hz, 6H), −0.18 (s, 3H), −0.41 (s, 3H); ³¹P NMR (121 MHz,DMSO-d₆) δ 72.91; LC/MS (ESI, m/z): [(M+1)]⁺=1324.5. Isomer A and isomerB were independently carried through the remainder of the synthesis(Step 5 through Step 10).

Step 5

O-(((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl)O-(2-cyanoethyl)((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-((((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)methyl)tetrahydrofuran-3-yl)phosphoramidothioate(144)

To a solution ofO-(((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl)O-(2-cyanoethyl)((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)phosphoramidothioate(143, 330 mg, 0.25 mmol, isomer A) in acetonitrile (2 mL) were added3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (150 mg, 0.50mmol) and pyridinium trifluoroacetate (66 mg, 0.34 mmol). The resultingsolution was stirred for 45 min at ambient temperature and was used inthe next step directly without workup: LC/MS (ESI, m/z):[(M+1)]⁺=1523.9.

Step 6

O-[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[({[(2S,3R,4R,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (145)

The above solution was sparged with hydrogen sulfide for 2 min followedby the addition of pyridinium trifluoroacetate (66 mg, 0.34 mmol). Theresulting solution was stirred for 30 min at ambient temperature andconcentrated under reduced pressure. The residue was applied onto areversed phase C18 column, eluting with 70%˜99% acetonitrile (25 min) inwater to afford the title compound 145 as a colorless solid (150 mg,40%, contains 5% 1H-phosphate byproduct): ¹H NMR (400 MHz, DMSO-d₆) δ12.10 (s, 1H), 11.46-11.41 (m, 1H), 11.20 (d, J=8.7 Hz, 1H), 8.72 (d,J=9.2 Hz, 1H), 8.68-8.57 (m, 1H), 8.21-8.14 (m, 1H), 8.05 (d, J=7.7 Hz,2H), 7.74-7.61 (m, 2H), 7.54 (dd, J=26.6, 7.7 Hz, 9H), 7.37 (t, J=7.6Hz, 7H), 7.26 (t, J=7.2 Hz, 3H), 6.25-6.18 (m, 1H), 6.09 (dd, J=6.0, 3.4Hz, 1H), 5.53 (d, J=10.8 Hz, 0.7H), 4.74 (s, 1.4H), 4.04 (q, J=7.1 Hz,2H), 3.73 (s, 2H), 3.28 (s, 2H), 2.93-2.73 (m, 6H), 2.54 (s, 2H),1.27-1.09 (m, 6H), 0.78-0.61 (m, 18H), −0.07-−0.19 (m, 9H), −0.36-−0.42(m, 3H); ³¹P NMR (162 MHz, DMSO-d₆) δ 73.27, 73.15, 73.09, 72.82; LC/MS(ESI, m/z): [(M+1)]⁺=1458.5. The same procedure was used on isomer B togenerate the other isomer as a colorless solid (190 mg, 45%): ¹H NMR(400 MHz, DMSO-d₆) δ 12.17-12.11 (m, 1H), 11.44 (d, J=3.5 Hz, 1H), 11.19(s, 1H), 8.80-8.63 (m, 2H), 8.67-8.57 (m, 1H), 8.14-8.02 (m, 3H),7.70-7.61 (m, 1H), 7.56 (dd, J=8.4, 7.0 Hz, 2H), 7.51-7.44 (m, 7H), 7.32(td, J=7.9, 1.9 Hz, 7H), 7.26-7.17 (m, 3H), 6.14 (ddd, J=5.7, 3.8, 2.1Hz, 2H), 5.65 (s, 1H), 4.74 (q, J=7.4, 5.7 Hz, 1H), 4.18 (d, J=9.7 Hz,1H), 4.09-3.90 (m, 6H), 3.85-3.76 (m, 1H), 3.21 (s, 1H), 2.94-2.75 (m,6H), 2.53 (m, 2H), 1.27-1.10 (m, 6H), 0.91-0.69 (m, 18H), 0.00-−0.09 (m,3H), −0.03-−0.11 (m, 3H), −0.14 (s, 3H), −0.36 (d, J=4.2 Hz, 3H); ³¹PNMR (162 MHz, DMSO-d₆) δ 73.31, 72.94, 72.70, 72.64; LC/MS (ESI, m/z):[(M+1)]⁺=1458.5.

Step 7

O-[(2S,3R,4R,5R)-3-[({[(2S,3R,4R,5R)-3-amino-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (146)

To a solution ofO-[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[({[(2S,3R,4R,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (145, 150 mg, 0.11 mmol) indichloromethane (3 mL) and water (10 mg, 0.5 mmol) was addeddichloroacetic acid (150 mg, 0.62 mmol). The resulting solution wasstirred for 20 min at ambient temperature and then quenched by theaddition of saturated aqueous solution of sodium bicarbonate (1.1 mL).The resulting mixture was extracted with dichloromethane (3×10 mL) andthe organic layers were combined and dried over anhydrous sodiumsulfate. After filtration, the filtrate was concentrated under reducedpressure and the residue was used in the next step without furtherpurification (146, 0.2 g, brown solid): LC/MS (ESI, m/z):[(M+1)]⁺=1215.4.

Step 8

N-{9-[(1R,6S,8R,9R,10R,15S,17R,18R)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-bis(2-cyanoethoxy)-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3,12-disulfanylidene-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(147)

To a solution of the aboveO-[(2S,3R,4R,5R)-3-[({[(2S,3R,4R,5R)-3-amino-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (146, 200 mg) in acetonitrile (30 mL)were added triethylamine (0.7 mL) and carbon tetrachloride (0.7 mL). Theresulting solution was stirred for 45 min at ambient temperature andconcentrated under reduced pressure to afford the crude title compound147 as a brown solid (200 mg) which was used in the next step directlywithout further purification: LC/MS (ESI, m/z): [(M+1)]⁺=1213.4.

Step 9

[(1R,6S,8R,9R,10R,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide,bis(methanaminium) (148)

The above crude product (147, 200 mg) was treated with methylamine (6mL, 30% ethanol solution, w/w) for 1.5 hour at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure and the residue was used in the next step directly withoutfurther purification (200 mg): LC/MS (ESI, m/z): [(M−2MeNH₂+1)]⁺=933.6.

Step 10

Diammonium[(1S,6S,8R,9R,10S,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide(72)

To a solution of the above crude compound 148 in pyridine (1.5 mL) wasadded triethylamine trihydrofluoride (1.2 mL) and triethylamine (0.4mL). The resulting solution was stirred for 2 hours at 60° C. Aftercooling down to ambient temperature, acetone (32 mL) was added toprecipitate the crude product which was purified using the followingconditions: column: Atlantis Prep T₃ OBD column, 19×250 mm, 10 um;Mobile Phase A: water (plus 20 mmol/L of NH₄HCO₃), Mobile Phase B: ACN;Flow rate: 20 mL/min; Gradient: 1% B to 14% B in 26 min; Detector:254/220 nm; to afford the mixed phosphate/thiophosphate 72-0A1 as acolorless solid (single isomer) (6.6 mg, retention time is 17.12 min):¹H NMR (400 MHz, D₂O+DMSO-d₆) δ 8.48 (br s, 0.6H), 8.24 (br s, 1.4H),8.12 (br s, 0.6H), 8.03 (br s, 0.4H), 5.95 (s, 1H), 5.76 (s, 1H),4.39-3.70 (m, 10H); ³¹P NMR (162 MHz, D₂O+DMSO-d₆) δ 56.24, 3.46, 2.01;LC/MS (ESI, m/z): [(M−2NH₃−1)]⁻=687.0. One isomer herein referred to72-0A2 as a colorless solid (6.3 mg, 9% over 4 steps, retention time is17.83 min): ¹H NMR (400 MHz, D₂O) δ 8.63 (s, 1H), 8.08 (s, 1H), 8.03 (s,1H), 6.07 (s, 1H), 5.88 (s, 1H), 4.36 (d, J=4.4 Hz, 1H), 4.29 (d, J=12.1Hz, 1H), 4.21 (d, J=11.9 Hz, 1H), 4.09 (d, J=11.2 Hz, 3H), 3.94 (ddt,J=26.6, 10.5, 5.1 Hz, 3H); ³¹P NMR (162 MHz, D₂O) δ 57.85, 54.61; LC/MS(ESI, m/z): [(M−2NH₃−1)]⁻=703.0. And the other isomer herein referred toas 72-0A3 as a colorless solid (6.8 mg, 10% over 4 steps, retention timeis 25.18 min): ¹H NMR (400 MHz, D₂O+DMSO-d₆) δ 8.39 (s, 1H), 8.21 (s,1H), 8.07 (s, 1H), 5.95 (s, 1H), 5.72 (s, 1H), 4.19-3.99 (m, 6H),3.91-3.78 (m, 4H); ³¹P NMR (162 MHz, D₂O+DMSO-d₆) δ 54.36; LC/MS (ESI,m/z): [(M−2NH₃−1)]⁻=703.0. FIG. 2A depicts a representation of compound72-0A3.

Steps 5-10 above were performed on the isomer B obtained in Step 4 togenerate the other two isomers, which were purified by Prep-HPLC withthe following conditions: Column: Atlantis Prep T₃ OBD column, 19×250mm, 10 um; Mobile Phase A: water (plus 20 mmol/L of NH₄HCO₃), MobilePhase B: ACN; Flow rate: 20 mL/min; Gradient: 0% B to 13% B in 21 min;Detector: 254/220 nm; One isomer herein referred to as 72-0B1 as acolorless solid (5.6 mg, 6% over 4 steps, retention time is 14.52 min):¹H NMR (400 MHz, D₂O) δ 8.65 (s, 1H), 8.20 (s, 1H), 8.09 (s, 1H), 6.09(s, 1H), 5.88 (s, 1H), 4.68 (m, 2H), 4.17-4.02 (m, 6H), 3.99-3.88 (m,1H), 3.87-3.68 (m, 1H); ³¹P NMR (162 MHz, D₂O) δ 57.91; LC/MS (ESI,m/z): [(M−2NH₃−1)]⁻=703.0. And the other isomer herein referred to as72-0B2 as a colorless solid (9.2 mg, 10% over 4 steps, retention time is18.50 min): ¹H NMR (400 MHz, D₂O) δ 8.41 (s, 1H), 8.21 (s, 1H), 8.09 (s,1H), 6.07 (s, 1H), 5.85 (s, 1H), 4.72 (d, J=4.8 Hz, 1H), 4.37-4.29 (m,2H), 4.20-3.81 (m, 7H); ³¹P NMR (162 MHz, D₂O) δ 57.94, 57.46, 54.46,50.95; LC/MS (ESI, m/z): [(M−2NH₃−1)]⁻=703.0. The isomers 72-0A2,72-0A3, 72-0B1, and 72-0B2 are believed to vary in stereochemicalconfiguration at the phosphorus atoms.

Diammonium(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate)

Step 1

N-(9-((2R,3R,4R,5S)-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-4-(tritylamino)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(149)

To a solution ofN-[9-[(2R,5S)-4-amino-3-[(tert-butyldimethylsilyl)oxy]-5-(hydroxymethyl)oxolan-2-yl]-9H-purin-6-yl]benzamide(INT-C, 1.20 g, 2.47 mmol) in pyridine (24 mL) were added triethylamine(0.69 mL, 4.95 mmol) and triphenylmethyl chloride (1.04 g, 3.70 mmol).The resulting mixture was stirred for 20 hours at ambient temperature.Upon completion, the reaction was quenched with methanol (10 mL). Theresulting solution was concentrated under reduced pressure and theresidue was purified by flash column chromatography, eluting with 1%methanol in dichloromethane to afford the title compound 149 as acolorless solid (0.80 g, 45%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.26-11.06(m, 1H), 8.69 (s, 1H), 8.68 (s, 1H), 8.02 (dd, J=7.1, 1.9 Hz, 2H),7.60-7.12 (m, 18H), 6.13 (d, J=3.3 Hz, 1H), 5.20-5.00 (m, 1H), 3.90-3.73(m, 1H), 3.64 (d, J=18.5 Hz, 2H), 3.34 (t, J=3.9 Hz, 1H), 3.16-3.03 (m,1H), 2.96 (d, J=6.3 Hz, 1H), 0.76 (s, 9H), −0.11 (s, 3H), −0.22 (s, 3H);LC/MS (ESI, m/z): [(M+1)]⁺=727.3.

Step 2

((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) diisopropylphosphoramidite (150)

To a solution ofN-(9-((2R,3R,4R,5S)-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-4-(tritylamino)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(149, 0.80 g, 1.10 mmol) in acetonitrile (20 mL) were added3-({bis[bis(propan-2-yl)amino]phosphanyl}oxy)propanenitrile (0.32 g,1.06 mmol) and pyridinium trifluoroacetate (0.66 g, 1.65 mmol). Theresulting solution was stirred for 1 hour at ambient temperature. Theresulting solution was used in the next step without work up: LC/MS(ESI, m/z): [(M+1)]⁺=927.4.

Step 3

((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) phosphonate (151)

To the above solution were added water (150, 0.20 g, 10.60 mmol) andpyridinium trifluoroacetate (0.50 g, 1.65 mmol). The resulting solutionwas stirred for 30 min at ambient temperature. Upon completion of thereaction, the resulting solution was applied onto a reversed phase C18column, eluting with 70%˜95% (25 min) acetonitrile in water to affordthe title compound 151 as a colorless solid (0.50 g, 54% for two steps):¹H NMR (300 MHz, DMSO-d₆) δ 11.18 (s, 1H), 8.68 (s, 1H), 8.46 (s, 1H),8.14-8.00 (m, 2H), 7.67-6.97 (m, 18H), 6.15 (d, J=3.4 Hz, 1H), 5.55 (s,1H), 4.41-4.20 (m, 1H), 4.20-4.10 (m, 1H), 4.08-3.89 (m, 3H), 3.77-3.55(m, 1H), 3.19-3.01 (m, 2H), 2.83 (dt, J=7.8, 5.8 Hz, 2H), 0.79 (s, 9H),−0.10 (d, J=5.1 Hz, 3H), −0.22 (s, 3H); ³¹P NMR (121 MHz, DMSO-d₆) δ9.62, 8.87; LC/MS (ESI, m/z): [(M+1)]⁺=844.3.

Step 4

((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-3-(tritylamino)tetrahydrofuran-2-yl)methyl (2-cyanoethyl)((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)phosphoramidate(152)

To a mixture of((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) phosphonate (151, 0.51 g, 0.60 mmol) andN-(9-((2R,3R,4R,5S)-4-amino-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamidein(INT-C, 0.29 g, 0.60 mmol) in acetonitrile (6 mL) were addedN,N-diisopropylethylamine (0.15 g, 1.20 mmol) and carbon tetrachloride(0.36 g, 2.40 mmol). The resulting mixture was stirred for 15 min atambient temperature. Upon completion, the resulting mixture was loadedon a reversed phase C18 column, eluting with 70%˜95% (25 min)acetonitrile in water to afford the title compound 152 as a colorlesssolid (0.53 g, 67%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.27-11.06 (m, 2H),8.79-8.68 (m, 2H), 8.68-8.56 (m, 2H), 8.08-7.86 (m, 4H), 7.62-7.04 (m,21H), 6.19-5.93 (m, 2H), 5.27-5.09 (m, 1H), 5.09-4.82 (m, 1H), 4.67-4.47(m, 1H), 4.29-4.10 (m, 2H), 4.06-3.96 (m, 2H), 3.90-3.80 (m, 2H),3.75-3.62 (m, 1H), 3.61-3.52 (m, 1H), 3.16-2.94 (m, 2H), 2.80-2.68 (m,2H), 0.87-0.69 (m, 18H), 0.00 (d, J=8.4 Hz, 3H), −0.06 (d, J=3.7 Hz,3H), −0.12 (d, J=4.8 Hz, 3H), −0.21 (d, J=13.6 Hz, 3H); ³¹P NMR (121MHz, DMSO-d₆) δ 8.80, 8.31; LC/MS (ESI, m/z): [(M+1)]⁺=1326.9.

Step 5

((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl)((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-((((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)methyl)tetrahydrofuran-3-yl)phosphoramidate(153)

To a solution of((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-3-(tritylamino)tetrahydrofuran-2-yl)methyl (2-cyanoethyl)((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydrofuran-3-yl)phosphoramidate(152, 0.53 g, 0.40 mmol) in acetonitrile (5 mL) were added3-([bis[bis(propan-2-yl)amino]phosphanyl]oxy)propanenitrile (0.24 g,0.80 mmol) and pyridinium trifluoroacetate (0.12 g, 0.60 mmol). Theresulting mixture was stirred for 50 min at ambient temperature and wasused in the next step directly.

Step 6

[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-[({[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2-cyanoethoxy)phosphoryl)amino]-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methyl-2-cyanoethylphosphorate (154)

To the above solution were added water (0.07 g, 4.0 mmol) and pyridiniumtrifluoroacetate (0.12 g, 0.60 mmol). The resulting solution was stirredfor 30 mins at ambient temperature. Upon completion, the resultingsolution was applied onto a reversed phase C18 column, eluting with70%˜95% (25 min) acetonitrile in water to afford the title compound 154as a colorless solid (0.50 g, 87% for two steps): ¹H NMR (300 MHz,DMSO-d₆) δ 11.24-11.08 (m, 2H), 8.76-8.45 (m, 4H), 8.05-7.97 (m, 4H),7.63-7.07 (m, 21H), 6.20-5.97 (m, 2H), 5.24-4.88 (m, 1H), 4.83-4.62 (m,1H), 4.43-3.91 (m, 10H), 3.94-3.81 (m, 1H), 3.15-2.97 (m, 2H), 2.92-2.72(m, 4H), 0.84-0.69 (m, 18H), 0.05-−0.04 (m, 4H), −0.07-−0.25 (m, 8H);³¹P NMR (121 MHz, DMSO-d₆) δ 9.90, 9.81, 9.28, 9.23, 8.77, 8.73, 8.10,8.07. LC/MS (ESI, m/z): [(M+1)]⁺=1444.5

Step 7

[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-[({[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2-cyanoethoxy)phosphoryl)amino]-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methyl-2-cyanoethylphosphor-nate (155)

To a solution of[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-[({[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2cyanoethoxy)phosphoryl)amino]-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methyl2-cyanoethyl phosphonate (154, 504 mg, 0.35 mmol) in dichloromethane (7mL) were added water (31.4 mg, 0.98 mmol) and dichloroacetic acid (447mg, 1.96 mmol). The resulting solution was stirred for 30 min at ambienttemperature. Upon completion, the reaction was quenched by the additionof saturated aqueous solution of sodium bicarbonate (30 mL). Theresulting mixture was extracted with dichloromethane (3×30 mL). Theorganic layers were combined and dried over anhydrous sodium sulfate.After filtration, the filtrate was concentrated under reduced pressureto afford the title compound 155, which was used in the next stepwithout further purification: LC/MS (ESI, m/z): [(M+1)]⁺=1201.4.

Step 8

N-{9-[(1R,6S,8R,9R,10R,15S,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-bis(2-cyanoethoxy)-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide156)

To a solution of the above crude compound 155 in acetonitrile (70 mL)were added triethylamine (1.4 mL) and carbon tetrachloride (1.4 mL). Theresulting solution was stirred for 10 min at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure to afford the crude title compound 156, which was used in thenext step without further purification.

Step 9

(1R,6S,8R,9R,10R,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate),bis(methanaminium) (157)

The above crude compound 156 was treated with a solution of methylaminein ethanol (14 mL, 30%, w/w) for 30 min at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure to afford the title compound 157 as a colorless solid, whichwas used in the next step without further purification.

Step 10

Diammonium(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)] octadec-ane-3,12-bis(olate) (73)

To a solution of the above crude compound 157 in pyridine (3.48 mL) wereadded triethylamine (1 mL) and triethylamine trihydrofluoride (2.8 g,17.4 mmol). The resulting solution was stirred for 2 hours at 60° C.After cooling down to ambient temperature, acetone (200 ml) was added toprecipitate the crude product which was purified by Prep-HPLC with thefollowing conditions: Column: Atlantis Prep T3 OBD Column, 19*250 mm, 10um; Mobile Phase A: Water (plus 20 mmol/L of NH₄HCO₃); Mobile Phase B:ACN; Flow rate: 20 mL/min; Gradient: 0% B to 15% B in 11 min; Detector:254/220 nm; to afford the title compound 73 as a colorless solid. (0.15g, 62% for 4 steps, retention time is 10.18 min): ¹H NMR (300 MHz, D₂O)δ 8.59-8.40 (m, 2H), 8.04-7.79 (m, 2H), 6.08 (d, J=1.0 Hz, 2H), 4.40 (d,J=3.9 Hz, 2H), 4.30-4.17 (m, 2H), 4.17-3.89 (m, 4H), 3.78-3.52 (m, 2H);³¹P NMR (121 MHz, D₂O) δ 6.23; LC/MS (ESI, m/z): [(M−2NH₃−1)]⁻=655.05.

Diammonium[(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide

Step 1 CN

((2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-((tert-butyldimethylsilyl)oxy)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl) diisopropylphosphoramidite (150)

To a solution ofN-(9-((2R,3R,4R,5S)-3-((tert-butyldimethylsilyl)oxy)-5-(hydroxymethyl)-4-(tritylamino)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(149, 1.40 g, 1.93 mmol) in acetonitrile (13.7 mL) were added3-({bis[bis(propan-2-yl)amino]phosphanyl}oxy)propanenitrile (1.40 ml,3.86 mmol) and pyridinium trifluoroacetate (0.56 g, 2.80 mmol). Theresulting solution was stirred for 1 hour at ambient temperature. Theresulting solution was used in the next step without work up: LC/MS(ESI, m/z): [(M+1)]⁺=927.4.

Step 2

O-[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[(triphenylmethyl)amino]oxolan-2-yl]methyl O-2-cyanoethyl phosphonothioate(158)

To the above solution was bubbled hydrogen sulfide gas for 1 minfollowed by the addition of pyridinium trifluoroacetate (0.56 g, 2.8mmol). The resulting solution was stirred for 30 min at ambienttemperature. Upon completion, the resulting solution was applied onto areversed phase C18 column, eluting with 75%˜99% (25 min) acetonitrile inwater to afford the title compound 158 as a colorless solid (1.12 g, 68%for two steps): ¹H NMR (300 MHz, DMSO-d₆) δ 11.20 (s, 1H), 8.71 (d,J=3.5 Hz, 1H), 8.48 (d, J=8.6 Hz, 1H), 8.05 (d, J=7.6 Hz, 2H), 7.66-7.08(m, 18H), 6.19 (t, J=3.1 Hz, 1H), 4.42-4.19 (m, 2H), 4.05-3.97 (m, 1H),3.78-3.66 (m, 1H), 3.36-3.21 (m, 2H), 3.23-3.03 (m, 2H), 2.95-2.75 (m,2H), 0.80 (s, 9H), −0.08 (d, J=5.5 Hz, 3H), −0.19 (d, J=2.2 Hz, 3H); ³¹PNMR (121 MHz, DMSO) δ 73.01, 72.43; LC/MS (ESI, m/z): [(M+1)]⁺=860.0

Step 3

N-{9-[(2R,3R,4R,5S)-5-{[({[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-2-(hydroxymethyl)oxolan-3-yl]amino}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)oxy]methyl}-3-[(tert-butyldimethylsilyl)oxy]-4-[(triphenylmethyl)amino]oxolan-2-yl]-9H-purin-6-yl}benzamide (159)

To a mixture of[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[(triphenylmethyl)amino]oxolan-2-yl]methyl-2-cyanoethylphosphonate(158, 1.12 g, 1.33 mmol) andN-9-[(2R,5S)-4-amino-3-[(tert-butyldimethylsilyl)oxy]-5-(hydroxylmethyl)oxolan-2-yl]-9H-purin-6-ylbenzamide (0.29 g, 1.33 mmol) inacetonitrile (13 mL) were added N,N-diisopropylethylamine (0.34 g, 2.66mmol) and carbon tetrachloride (0.79 g, 5.32 mmol). The resultingsolution was stirred for 15 min at ambient temperature. Upon completion,the resulting mixture was applied onto a reversed phase C18 column,eluting with 75%˜99% (25 min) acetonitrile in water to afford the titlecompound 159 as a colorless solid (1.1 g, 62%): ¹H NMR (300 MHz,DMSO-d₆) δ 11.22-11.01 (m, 2H), 8.73-8.34 (m, 4H), 8.05-7.94 (m, 4H),7.63-7.05 (m, 21H), 6.21-5.96 (m, 2H), 5.69-5.45 (m, 1H), 5.13 (dt,J=25.9, 5.1 Hz, 1H), 4.61-4.51 (m, 1H), 4.40-4.20 (m, 1H), 4.18-3.94 (m,5H), 3.79-3.70 (m, 1H), 3.66-3.46 (m, 1H), 3.16-2.94 (m, 2H), 2.88-2.75(m, 2H), 0.83-0.64 (m, 18H), 0.07-−0.04 (m, 3H), −0.07-−0.28 (m, 9H);³¹P NMR (121 MHz, DMSO) δ 73.32, 72.28; LC/MS (ESI, m/z):[(M+1)]⁺=1342.5.

Step 4

N-{9-[(2R,3R,4R,5S)-5-({[(R)-{[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-[({[bis(propan-2-yl)amino](2-cyanoethoxy)phosphanyl}oxy)methyl]-4-[(tert-butyldimethylsilyl)oxy]oxolan-3-yl]amino}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl]oxy}methyl)-3-[(tert-butyldimethylsilyl)oxy]-4-[(triphenylmethyl)amino]oxolan-2-yl]-9H-purin-6-yl}benzamide(160)

To a solution ofN-{9-[(2R,3R,4R,5S)-5-{[({[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldi-methylsilyl)oxy]-2-(hydroxymethyl)oxolan-3-yl]amino}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)oxy]methyl}-3-[(tert-butyldimethylsilyl)oxy]-4-[(triphenylmethyl)amino]oxolan-2-yl]-9H-purin-6-yl}benzamide(159, 1.1 g, 0.82 mmol) in acetonitrile (3.3 mL) were added3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (0.49 g, 1.64mol) and pyridinium trifluoroacetate (0.24 g, 1.23 mmol). The resultingmixture was stirred for 50 min at ambient temperature and was used inthe next step directly.

O-[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-[({[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanvlidene-λ⁵-phosphanyl)amino]-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (161)

To the above solution was bubbled with hydrogen sulfide for 1 minfollowed by the addition of pyridinium trifluoroacetate (0.24 g, 1.23mmol). After stirring for 30 min at ambient temperature, the resultingsolution was applied onto a reversed phase C18 column, eluting with75%˜99% (25 min) acetonitrile in water to afford two isomers of thetitle compound as a colorless solid: isomer A (faster eluting part,eluted with 95% acetonitrile) (530 mg, 44% for two steps, contains 10%phosphate byproduct): ¹H NMR (300 MHz, DMSO-d₆) δ 11.32-10.99 (m, 2H),8.78-8.43 (m, 4H), 8.10-7.95 (m, 4H), 7.77-6.95 (m, 21H), 6.25-5.98 (m,2H), 5.64-5.30 (m, 1H), 4.82-4.42 (m, 1H), 4.46-3.85 (m, 10H), 3.85-3.61(m, 1H), 3.61-3.37 (m, 1H), 3.23-2.97 (m, 2H), 2.92-2.66 (m, 4H),0.89-0.56 (m, 18H), −0.07-−0.27 (m, 12H); ³¹P NMR (121 MHz, DMSO) δ74.19, 74.06, 73.96, 73.46, 73.37, 73.31, 73.27, 72.79, 9.69, 9.15,5.34, 4.70, 1.38, −1.11, −1.22, −1.67, −1.92; LC/MS (ESI, m/z):[(M+1)]⁺=1475.4. And isomer B (slower eluting part, eluted with 99%acetonitrile) (560 mg, 46% for two steps, contains 10% phosphatebyproduct): ¹H NMR (300 MHz, DMSO-d₆) δ 11.26-10.97 (m, 2H), 8.72-8.38(m, 4H), 8.12-7.90 (m, 4H), 7.67-7.00 (m, 21H), 6.20-5.87 (m, 2H),4.80-4.55 (m, 1H), 4.36-3.75 (m, 10H), 3.62-3.44 (m, 1H), 3.31-3.13 (m,1H), 3.13-2.91 (m, 2H), 2.89-2.62 (m, 4H), 0.97-0.57 (m, 18H),0.10-−0.01 (m, 3H), −0.04-−0.24 (m, 9H); ³¹P NMR (121 MHz, DMSO) δ114.61, 73.39, 73.00, 72.66, 72.26, 72.22, 72.07, 71.94, 9.83, 9.24,5.35, 4.70, 1.37, −1.12, −1.18; LC/MS (ESI, m/z): [(M+1)]⁺=1475.4.Isomer A and isomer B were independently carried through the remainderof the synthesis (Step 6 through Step 9).

Step 6

O-[(2S,3R,4R,5R)-3-[({[(2S,3R,4R,5R)-3-amino-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (162)

To a solution ofO-[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-[({[(2S,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-[(tert-butyldimethylsilyl)oxy]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]-4-[(tert-butyldimethylsilyl)oxy]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (161, 530 mg, 0.36 mmol, isomer A fromprevious step) in dichloromethane (7.2 mL) were added dichloroaceticacid (460 mg, 3.59 mmol) and water (32 mg, 1.80 mmol). The resultingsolution was stirred for 30 min at ambient temperature. Upon completion,the reaction was quenched by the addition of saturated aqueous sodiumbicarbonate (30 mL). The resulting mixture was extracted withdichloromethane (3×30 mL). The organic layers were combined and driedover anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure to afford the title compound 162,which was used in the next step without further purification.

Step 7

N-{9-[(1R,6S,8R,9R,10R,15S,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-bis(2-cyanoethoxy)-3,12-disulfanylidene-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(163)

To a solution of the above crude compound 162 in acetonitrile (72 mL)were added triethylamine (1.44 mL) and carbon tetrachloride (1.44 mL).The resulting solution was stirred for 10 min at ambient temperature.Upon completion, the resulting solution was concentrated under reducedpressure to afford the crude title compound 163, which was used in thenext step without further purification.

Step 8

[(1R,6S,8R,9R,10R,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide,bis(methanaminium) 164)

The above crude compound was treated with a solution of methylamine inethanol (163, 14.4 mL, 30%, w/w) for 30 min at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure to afford the title compound 164 as a colorless solid, whichwas used in the next step without further purification.

Step 9

Diammonium[(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide (74)

To a solution of the above crude compound 164 in pyridine (3.6 mL) wereadded triethylamine (1 mL) and triethylamine trihydrofluoride (2.89 g,17.95 mmol). The resulting solution was stirred for 2 hours at 60° C.After cooling down to ambient temperature, acetone (200 mL) was added toprecipitate the crude product which was purified by Prep-HPLC with thefollowing conditions: Column: XBridge Prep C18 OBD Column 19*150 mm, 5um; Mobile Phase A: Water (plus 20 mmol/L of NH₄HCO₃); Mobile Phase B:ACN; Flow rate: 20 mL/min; Gradient: 1% B to 15% B in 11 min; Detector:254/220 nm; to afford one isomer (faster eluting peak, retention time is7.52 min, herein referred to 74-0A1) as a colorless solid (40.2 mg,16.3% for 4 step): ¹H NMR (300 MHz, D₂O) δ 8.49 (s, 1H), 8.41 (s, 1H),8.14-8.02 (m, 1H), 7.96-7.86 (m, 1H), 6.08 (s, 1H), 6.05 (s, 1H), 4.82(d, J=4.1 Hz, 1H), 4.40-4.28 (m, 2H), 4.26-3.93 (m, 5H), 3.75 (d, J=10.6Hz, 2H); ³¹P NMR (121 MHz, D₂O) δ 57.74, 54.73; LC/MS (ESI, m/z):[(M−2NH₃−1)]⁻=686.9. And the other isomer (slower eluting peak,retention time is 9.77 min, here referred to 74-0A2) as a colorlesssolid: ¹H NMR (300 MHz, D₂O+DMSO-d₆) δ 8.50 (s, 2H), 8.02 (s, 2H), 6.09(s, 2H), 4.50-4.27 (m, 4H), 4.13 (d, J=10.5 Hz, 2H), 3.98 (dd, J=11.9,5.4 Hz, 2H), 3.85 (s, 2H); ³¹P NMR (121 MHz, D₂O+DMSO-d₆) δ 52.39; LC/MS(ESI, m/z): [(M−2NH₃−1)]⁻=686.9.

Steps 6-9 above were performed on the isomer B obtained in Step 5 togenerate the other two isomers and a mixed phosphate/thiophosphate: Theywere purified by Prep-HPLC with the following conditions: XBridge PrepC18 OBD Column 19*150 mm, 5 um; Mobile Phase A: water (plus 20 mmol/L ofNH₄HCO₃); Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient: 1% B to40% B in 10 min; Detector: 254/220 nm; to afford the mixed phosphatethiophosphate diammonium salt 74-0B0

(single isomer)[(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-dihydroxy-12-oxido-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanideas a colorless solid (5.6 mg, retention time is 4.57 min): ¹H NMR (400MHz, D₂O) δ 8.72-8.49 (m, 2H), 8.16-7.95 (m, 2H), 6.24-6.09 (m, 2H),4.84 (q, J=5.8, 5.0 Hz, 1H), 4.48-4.38 (m, 1H), 4.33-4.21 (m, 2H),4.21-4.02 (m, 4H), 3.94-3.72 (m, 2H); ³¹P NMR (162 MHz, D₂O) δ 57.80,6.25; LC/MS (ESI, m/z): [(M−2NH₃−1)]⁻=670.9. And one isomer herereferred to 74-0B1 as a colorless solid (16.4 mg, 6% over 4 steps,retention time is 5.23 min): ¹H NMR (300 MHz, DMSO-d₆) δ 8.64 (s, 2H),8.13 (s, 2H), 5.97 (s, 2H), 4.37-4.29 (m, 2H), 4.11-4.00 (m, 2H),4.02-3.88 (m, 4H); ³¹P NMR (121 MHz, DMSO) δ 57.54. LC/MS (ESI, m/z):[(M−2NH₃−1)]⁻=686.9. The other isomer here referred to as 74-0B2 as acolorless solid (35 mg, 13% over 4 steps, retention time is 8.80 min),which has the same characteristics as 74-0A1. The isomers 74-0A1,74-0A2, 74-0B1, and 74-0B2 are believed to vary in stereochemicalconfiguration at the phosphorus atoms.

Diammonium(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate)

Step 1

((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl)((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)phosphoramidate(165)

To a mixture of[(2S,3R,4R,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3-[(triphenylmethyl)amino]oxolan-2-yl]methyl2-cyanoethyl phosphonate (1.0 g, 1.21 mmol) andN-(9-((2R,3R,4R,5S)-4-amino-3-(tert-butyldimethylsilyloxy)-5-(hydroxymethyl)-tetrahydrofuran-2-yl)-6-oxo-6,9-dihydro-H-purin-2-yl)isobutyramide(133, 0.56 g, 1.21 mmol) in acetonitrile (12 mL) were addedN,N-diisopropylethylamine (0.38 mL, 2.42 mmol) and carbon tetrachloride(0.47 mL, 4.84 mmol). The resulting mixture was stirred for 30 min atambient temperature. Upon completion, the resulting mixture was appliedonto a reversed phase C18 column, eluting with 70%˜95% (25 min)acetonitrile in water to afford the title compound 165 as a colorlesssolid (440 mg, 43%): ¹H NMR (400 MHz, DMSO-d₆) δ 12.12 (s, 2H), 11.46(t, J=11.1 Hz, 2H), 8.23 (s, 1H), 8.18 (s, 1H), 7.48 (dd, J=6.3, 2.9 Hz,6H), 7.40-7.31 (m, 6H), 7.25 (dt, J=9.4, 7.5 Hz, 3H), 6.22 (dd, J=17.5,6.5 Hz, 1H), 5.77 (dd, J=26.0, 4.5 Hz, 1H), 5.19-4.88 (m, 2H), 4.57-4.45(m, 1H), 4.35 (d, J=6.9 Hz, 1H), 4.19-3.39 (m, 6H), 3.27 (dd, J=17.9,2.3 Hz, 1H), 2.89-2.70 (m, 5H), 1.13 (dt, J=6.6, 3.2 Hz, 12H), 0.72 (d,J=32.6 Hz, 18H), 0.05-−0.22 (m, 9H), −0.43 (d, J=19.3 Hz, 3H); ³¹P NMR(162 MHz, DMSO-d₆) δ 8.98, 8.82; LC/MS (ESI, m/z): [(M+1)]⁺=1308.5.

Step 2

((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl(2-cyanoethyl)((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-((((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)methyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)phosphoramidate(166)

To a solution of((2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-3-(tritylamino)-tetrahydrofuran-2-yl)methyl2-cyanoethyl(2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-2-(hydroxymethyl)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-tetrahydrofuran-3-ylphosphoramidate(165, 1.0 g, 0.77 mmol) were added3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (224 mg, 0.74mmol) and pyridinium trifluoroacetate (224 mg, 1.16 mmol). The resultingmixture was stirred for 45 min at ambient temperature and was used inthe next step directly without further purification.

Step 3

((2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-3-(tritylamino)-tetrahydrofuran-2-yl)methyl2-cyanoethyl(2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-2-(((2-cyanoethoxy)hydrophosphoryloxy)methyl)-5-(2-isobutyramido-6-oxo-16-dihydropurin-9-yl)-tetrahydrofuran-3-ylphosphoramidate(167)

To the above solution were added water (144 mg, 7.99 mmol) andpyridinium trifluoroacetate (224 mg, 1.16 mmol). The resulting solutionwas stirred for 45 min at ambient temperature. Upon completion, theresulting solution was applied onto a reversed phase C18 column, elutingwith 70%˜95% (25 min) acetonitrile in water to afford the title compound167 as a colorless solid (800 mg, 73%): ¹H NMR (400 MHz, DMSO-d₆) δ12.07 (t, J=8.4 Hz, 2H), 11.40 (s, 2H), 8.23-8.03 (m, 2H), 7.51-7.15 (m,15H), 6.17 (dd, J=12.4, 6.4 Hz, 1H), 5.80-5.65 (m, 2H), 4.66-4.55 (m,1H), 4.25 (d, J=7.0 Hz, 1H), 4.19-3.94 (m, 5H), 3.96-3.76 (m, 3H), 3.60(s, 1H), 3.41 (dq, J=18.4, 6.9 Hz, 1H), 3.22 (d, J=16.2 Hz, 1H), 2.84(q, J=6.0 Hz, 5H), 1.09 (dt, J=6.8, 3.4 Hz, 12H), 0.73 (d, J=2.3 Hz,15H), 0.63 (d, J=1.9 Hz, 3H), −0.08 (d, J=2.3 Hz, 2H), −0.12-−0.27 (m,7H), −0.40-−0.51 (m, 3H); ³¹P NMR (162 MHz, DMSO-d₆) δ 9.75, 9.71, 9.23,9.14, 8.49, 8.34; LC/MS (ESI, m/z): [(M+1)]⁺=1407.5.

Step 4

((2S,3R,4R,5R)-3-amino-4-(tert-butyldimethylsilyloxy)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-tetrahydrofuran-2-yl)methyl2-cyanoethyl(2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-2-(((2-cyanoethoxy)hydrophosphoryloxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-tetrahydrofuran-3-ylphosphoramidate(168)

To a solution of((2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-3-(tritylamino)-tetrahydrofuran-2-yl)methyl2-cyanoethyl(2S,3R,4R,5R)-4-(tert-butyldimethylsilyloxy)-2-(((2-cyanoethoxy)hydrophosphoryloxy)methyl)-5-(2-isobutyramido-6-oxo-1,6-dihydropurin-9-yl)-tetrahydrofuran-3-ylphosphoramidate(167, 800 mg, 0.57 mmol) in dichloromethane (15 mL) were added water(0.51 mL) and dichloroacetic acid (0.47 mL, 5.7 mmol). The resultingsolution was stirred for 10 min at ambient temperature, Upon completion,the reaction was quenched by the addition of saturated aqueous solutionof sodium bicarbonate (30 mL). The resulting mixture was extracted withdichloromethane (3×30 mL). The organic layers were combined and driedover anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure to afford the title compound 168,which was used in the next step without further purification: LC/MS(ESI, m/z): [(M+1)]⁺=1165.4.

Step 5

N-{9-[(1R,6S,8R,9R,10R,15S,17R,18R)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-bis(2-cyanoethoxy)-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-8-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-methylpropanamide(169)

To a solution of the above crude compound 168 in acetonitrile (80 mL)were added triethylamine (1.4 mL) and carbon tetrachloride (1.4 mL). Theresulting solution was stirred for 10 min at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure to afford the crude title compound 169, which was used in thenext step without further purification: LC/MS (ESI, m/z):[(M+1)]⁺=1163.5.

Step 6

(1R,6S,8R,9R,10R,15S,17R,18R)-8,17-bis(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-dihydroxy-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-dione(170)

The above crude compound was treated with a solution of methylamine inethanol (14 mL, 30%, w/w) for 30 min at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure to afford the title compound 170 as a colorless solid, whichwas used in the next step without further purification: LC/MS (ESI,m/z): [(M−2MeNH₂+1)]⁺=917.7.

Step 7

diammonium(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate)(75)

To a solution of the above crude compound in pyridine (3 mL) were addedtriethylamine (0.42 mL) and triethylamine trihydrofluoride (1.18 g,14.00 mmol). The resulting solution was stirred for 2 hours at 60° C.After cooling down to ambient temperature, acetone (30 mL) was added toprecipitate the crude product which was purified by Prep-HPLC with thefollowing conditions: Column: Atlantis Prep T3 OBD Column, 19*250 mm, 10um; Mobile Phase A: water (plus 20 mmol/L of NH₄HCO₃), Mobile Phase B:ACN; Flow rate: 20 mL/min; Gradient: 0% B to 12% B in 10 min; Detector:254/220 nm; Retention time: 9.35 min; to afford the title compound 75 asa colorless solid (50.7 mg, 15%): ¹H NMR (400 MHz, D₂O) δ 8.06 (s, 2H),5.74 (s, 2H), 4.47 (s, 2H), 4.19 (d, J=11.8 Hz, 2.3H), 4.09-3.98 (m,6.5H); ³¹P NMR (162 MHz, D₂O) δ 6.97; LC/MS (ESI, m/z):[(M−2NH₃−1)]⁻=687.1.

Diammonium[(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-9,18-dihydroxy-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12)⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide

Step 1

O-(((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl)O-(2-cyanoethyl)((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)phosphoramidothioate(171)

To a mixture of[(3S,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3-[(triphenylmethyl)amino]oxolan-2-yl]methyl2-cyanoethyl sulfanylidenephosphonite (142, 1.20 g, 1.42 mmol) andN-9-[(2R,4S)-4-amino-3-[(tert-butyldimethylsilyl)oxy]-5-(hydroxymethyl)oxolan-2-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl-2-methylpropanamide(0.67 g, 1.42 mmol) in acetonitrile (15 mL) were addedN,N-diisopropylethylamine (0.48 mL, 2.84 mmol) and carbon tetrachloride(0.60 mL, 5.68 mmol). The resulting solution was stirred for 30 min atambient temperature. Upon completion, the resulting mixture was appliedonto a reversed phase C18 column, eluting with 75%˜99% (25 min)acetonitrile in water to afford the title compound 171 (two isomers:isomer A and isomer B) as a colorless solid: Isomer A (faster elutingpart, eluted with 95% acetonitrile) (325 mg, 18%): ¹H NMR (400 MHz,DMSO-d₆) δ 12.10 (s, 2H), 11.45 (d, J=15.3 Hz, 2H), 8.19 (d, J=2.6 Hz,2H), 7.54-7.47 (m, 6H), 7.37 (t, J=7.7 Hz, 6H), 7.31-7.22 (m, 3H), 6.23(d, J=6.6 Hz, 1H), 5.75 (d, J=4.9 Hz, 1H), 5.33 (dd, J=11.2, 7.3 Hz,1H), 5.00 (t, J=5.3 Hz, 1H), 4.82 (s, 1H), 4.52 (t, J=5.3 Hz, 1H), 4.05(q, J=9.8 Hz, 1H), 3.98-3.80 (m, 5H), 3.69 (t, J=9.3 Hz, 1H), 3.55 (dd,J=11.5, 5.0 Hz, 1H), 3.46-3.33 (m, 1H), 3.27 (d, J=2.2 Hz, 1H), 2.79(dtd, J=19.7, 6.3, 4.4 Hz, 5H), 1.25 (d, J=3.1 Hz, 2H), 1.17-1.09 (m,10H), 0.75 (s, 9H), 0.67 (s, 9H), −0.09-−0.24 (m, 9H), −0.39 (s, 3H);³¹P NMR (162 MHz, DMSO-d₆) δ 73.63; LC/MS (ESI, m/z): [(M+1)]⁺=1306.5.And isomer B (slower eluting part, eluted with 99% acetonitrile) (452mg, 25%): ¹H NMR (400 MHz, DMSO-d₆) δ 12.14 (d, J=7.5 Hz, 2H), 11.48 (d,J=18.3 Hz, 2H), 8.18 (s, 1H), 8.11 (s, 1H), 7.51-7.44 (m, 6H), 7.33 (t,J=7.7 Hz, 6H), 7.23 (dd, J=8.2, 6.4 Hz, 3H), 6.14 (d, J=5.7 Hz, 1H),5.82 (d, J=4.7 Hz, 1H), 5.56 (d, J=9.9 Hz, 1H), 5.11 (t, J=5.2 Hz, 1H),4.50 (t, J=5.2 Hz, 1H), 4.22 (s, 1H), 3.95 (dd, J=13.6, 7.2 Hz, 6H),3.79 (d, J=9.4 Hz, 1H), 3.64 (s, 1H), 3.48 (s, 1H), 3.21 (s, 1H),2.90-2.75 (m, 4H), 1.14 (ddd, J=10.0, 6.8, 1.6 Hz, 12H), 0.75 (d, J=5.0Hz, 18H), −0.05 (s, 3H), −0.14 (d, J=2.6 Hz, 6H), −0.35 (s, 3H); ³¹P NMR(162 MHz, DMSO-d₆) δ 73.21; LC/MS (ESI, m/z): [(M+1)]⁺=1306.5. Isomer Aand isomer B were independently carried through the remainder of thesteps (Step 2 through Step 7).

Step 2

O-(((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl)O-(2-cyanoethyl)((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-((((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)methyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)phosphoramidothioate(172)

To a solution ofO-(((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)-3-(tritylamino)tetrahydrofuran-2-yl)methyl)O-(2-cyanoethyl)((2S,3R,4R,5R)-4-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)-5-(2-isobutyramido-6-oxo-1H-purin-9(6H)-yl)tetrahydrofuran-3-yl)phosphoramidothioate(171 isomer A, 325 mg, 0.25 mmol) in acetonitrile (2 mL) were added3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (150 mg, 0.50mol) and pyridinium trifluoroacetate (72 mg, 0.37 mmol). The resultingmixture was stirred for 50 min at ambient temperature and was used inthe next step directly.

Step 3

O-[(2S,3R,4R,5R)-4-[(tert-butyldimethylsilyl)oxy]-3-[({[(2S,3R,4R,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (173)

To the above solution was bubbled with hydrogen sulfide for 1 minfollowed by the addition of pyridinium trifluoroacetate (72 mg, 0.37mmol). After stirring for 30 min at ambient temperature, the resultingsolution was applied onto a reversed phase C18 column, eluting with75%˜99% (25 min) acetonitrile in water to afford the desired compound asa colorless solid (278 mg, 80% for two steps): LC/MS (ESI, m/z):[(M+1)]⁺=1439.9.

Step 4

O-[(2S,3R,4R,5R)-3-[({[(2S,3R,4R,5R)-3-amino-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (174)

To a solution ofO-[(2S,3R,4R,5R)-4-[(tert-butyldimethylsilyl)oxy]-3-[({[(2S,3R,4R,5R)-4-[(tert-butyldimethylsilyl)oxy]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3-[(triphenylmethyl)amino]oxolan-2-yl]methoxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]-5-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]oxolan-2-yl]methylO-2-cyanoethyl phosphonothioate (173, 278 mg, 0.19 mmol) indichloromethane (4 mL) were added dichloroacetic acid (247 mg, 1.10mmol) and water (17.4 mg, 0.97 mmol). The resulting solution was stirredfor 20 min at ambient temperature. Upon completion, the reaction wasquenched by the addition of saturated aqueous solution of sodiumbicarbonate (30 mL). The resulting mixture was extracted withdichloromethane (3×30 mL). The organic layers were combined and driedover anhydrous sodium sulfate. After filtration, the filtrate wasconcentrated under reduced pressure to afford the title compound 174,which was used in the next step without further purification.

Step 5

N-{9-[(1R,6S,8R,9R,10R,15S,17R,18R)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-bis(2-cyanoethoxy)-17-[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3,12-disulfanylidene-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-8-yl]-6-oxo-6,9-dihydro-1H-purin-2-yl}-2-methylpropanamide(175)

To a solution of the above crude compound 174 in acetonitrile (38.6 mL)were added triethylamine (0.77 mL) and carbon tetrachloride (0.77 mL).The resulting solution was stirred for 10 min at ambient temperature.Upon completion, the resulting solution was concentrated under reducedpressure to afford the crude title compound 175, which was used in thenext step without further purification.

Step 6

[(1R,6S,8R,9R,10R,15S,17R,18R)-8,17-bis(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-9,18-bis[(tert-butyldimethylsilyl)oxy]-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide,bis(methanaminium) (176)

The above crude compound 175 was treated with a solution of methylaminein ethanol (14.4 mL, 30%, w/w) for 30 min at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure to afford the title compound 176 as a colorless solid, whichwas used in the next step without further purification.

Step 7

Diammonium[(1S,6S,8R,9R,10S,15S,17R,18R)-9,18-dihydroxy-8,17-bis[2-(2-methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-yl]-3,12-dioxo-12-sulfanidyl-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide (76)

To a solution of the above crude compound 176 in pyridine (2.76 mL) wereadded triethylamine (0.41 mL) and triethylamine trihydrofluoride (2.2mL). The resulting solution was stirred for 2 hours at 60° C. Aftercooling down to ambient temperature, acetone (55 mL) was added toprecipitate the crude product which was purified by Prep-HPLC with thefollowing conditions: column: Atlantis Prep T₃ OBD column, 19×250 mm, 10um; Mobile Phase A: water (plus 10 mmol/L of NH₄HCO₃); Mobile Phase B:ACN; Flow rate: 20 mL/min; Gradient: 1% B to 12% B in 26 min; Detector:254/220 nm; to afford the mixed phosphate/thiophosphate diammonium salt[(1S,6S,8R,9R,10S,15S,17R,18R)-8,17-bis(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-9,18-dihydroxy-12-oxido-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide(the first eluting peak, retention time is 14.65 min, herein referred to76-0A1) as a colorless solid (3.6 mg, 1.4%): ¹H NMR (400 MHz, D₂O) δ7.97 (d, J=11.2 Hz, 2H), 5.79 (d, J=5.1 Hz, 2H), 4.51 (s, 1H), 4.44 (s,1H), 4.36 (d, J=12.0 Hz, 1H), 4.29-3.98 (m, 7H); ³¹P NMR (162 MHz, D₂O)δ 57.92, 56.01, 54.65, 7.09; LC/MS (ESI, m/z): [(M−1)]=703.0. One isomer(the second eluting peak, retention time is 14.97 min, herein referredto 76-0A2) as a colorless solid (14.6 mg, 5.7%): ¹H NMR (400 MHz, D₂O) δ8.28 (s, 1H), 8.10 (s, 1H), 5.94 (s, 2H), 4.41-4.28 (m, 2H), 4.23-3.91(m, 8H); ³¹P NMR (162 MHz, D₂O) δ 58.10, 54.47; LC/MS (ESI, m/z):[(M−1)]⁻=718.95. And the other isomer (the third eluting peak, retentiontime is 22.75 min, herein referred to 76-0A3) as a colorless solid (22.8mg, 8.9%): ¹H NMR (400 MHz, D₂O) δ 10.33 (s, 2H), 7.96 (s, 2H), 6.36 (m,4H), 6.24 (m, 2H), 6.03 (m, 3H); ³¹P NMR (162 MHz, D₂O) δ 56.81; LC/MS(ESI, m/z): [(M−1)]⁻=718.9.

Steps 2-7 were performed on the isomer B obtained in step 1 to generatethe last desired product, herein referred to as 76-0B1, which waspurified by Prep-HPLC with the following conditions: column: XBridgePrep C18 OBD column 19×150 mm, 5 um; Mobile Phase A: water (plus 20mmmol/L of NH₄HCO₃); Mobile Phase B: ACN; Flow rate: 20 mL/min;Gradient: 1% B to 9% B in 8 min; Detector: 254/220 nm; retention time is6.85 min (7.3 mg, yield for 5 steps is 3.8%): ¹H NMR (300 MHz, D₂O) δ8.24 (s, 1H), 8.04 (s, 1H), 5.88 (s, 2H), 4.37-4.21 (m, 2H), 4.20-3.86(m, 8H); ³¹P NMR (121 MHz, D₂O) δ 58.03, 54.44; LC/MS (ESI, m/z):[(M−1)]⁻=719.0. The isomers 76-0A2, 76-0A3, and 76-0B1 are believed tovary in stereochemical configuration at the phosphorus atoms.

Diammonium[(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-difluoro-3,12-dioxo-12-sulfanidyl-2,7,11,16-tetraoxa-4,13-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide

Step 1

(2R,3R,4R,5S)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite 177

To a solution ofN-(9-((2S,3R,4R,5R)-3-fluoro-4-hydroxy-5-((tritylamino)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(INT-E, 0.80 g, 1.31 mmol) in acetonitrile (5 mL) were added pyridiniumtrifluoroacetate (0.38 g, 1.91 mmol) and3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (0.78 g, 2.60mmol). The resulting solution was stirred for 2 hours at ambienttemperature. Upon completion, the resulting solution was applied onto areversed phase C18 column, eluting with 70%˜95% (25 min) acetonitrile inwater to afford the title compound 177 as a colorless solid (0.8 g,75%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.25 (s, 1H), 8.67 (d, J=2.5 Hz, 1H),8.40-8.32 (m, 1H), 8.07 (d, J=7.5 Hz, 2H), 7.74-7.08 (m, 18H), 6.44(ddd, J=19.5, 11.7, 2.9 Hz, 1H), 5.89 (dq, J=52.9, 3.8 Hz, 1H),5.38-5.09 (m, 1H), 4.36-4.20 (m, 1H), 3.97-3.78 (m, 1H), 3.78-3.48 (m,3H), 3.09-2.63 (m, 3H), 2.43 (d, J=5.3 Hz, 1H), 1.31-0.96 (m, 12H); ¹⁹FNMR (282 MHz, DMSO-d₆) δ −200.77, −200.79, −201.31, −201.36; ³¹P NMR(121 MHz, DMSO-d₆) δ 149.59, 149.50, 149.45; LC/MS (ESI, m/z):[(M+1)]⁺=787.0.

Step 2

O-((2R,3R,4R,5S)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl)O-(2-cyanoethyl)phosphonothioate (178)

To a mixture of(2R,3R,4R,5S)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl(2-cyanoethyl) diisopropylphosphoramidite (177, 0.80 g, 0.98 mmol) and1H-1,2,3,4-tetrazole (69 mg, 0.98 mmol) in dry acetonitrile (5 mL) wasbubbled hydrogen sulfide for 1 min at ambient temperature. The resultingsolution was sealed and stirred for another 1 hour at ambienttemperature. Upon completion, the resulting solution was applied onto areversed phase C18 column, eluting with 70%˜95% (25 min) acetonitrile inwater to afford the title compound 178 as a colorless solid (0.70 g,95%): ¹H NMR (400 MHz, DMSO-d₆) δ 11.26 (s, 1H), 8.66-8.65 (m, 1H), 8.41(s, 1H), 8.08-8.04 (m, 2H), 7.70-7.20 (m, 18H), 6.46 (dd, J=18.2, 2.6Hz, 1H), 6.16-5.24 (m, 2H), 4.65-4.50 (m, 1H), 4.37-3.83 (m, 4H),3.11-2.93 (m, 1H), 2.93-2.72 (m, 3H); ¹⁹F NMR (376 MHz, DMSO-d₆) δ−201.20, −203.22; ³¹P NMR (162 MHz, DMSO-d₆) δ −2.64; LC/MS (ESI, m/z):[(M+1)]⁺=748.0.

Step 3

O-((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl)O-(2-cyanoethyl)(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)phosphoramidothioate(179)

To a solution ofO-((2R,3R,4R,5S)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl)O-(2-cyanoethyl)phosphonothioate (178, 0.70 g, 0.94 mmol) in N-methyl pyrrolidone (5 mL)were added N,N-diisopropylethylamine (0.48 g, 3.76 mmol),N-(9-((2S,3R,4R,5R)-5-(aminomethyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(349 mg, 0.94 mmol) and carbon tetrachloride (0.58 mg, 3.76 mmol) atambient temperature. The resulting solution was stirred for 2 hours atambient temperature. Upon completion, the resulting solution was appliedonto a reversed phase C18 column, eluting with 70%˜95% (25 min)acetonitrile in water to afford the title compound 179 as a colorlesssolid (0.70 g, 67%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.26 (s, 2H), 8.78 (d,J=4.0 Hz, 1H), 8.74-8.59 (m, 2H), 8.15-7.96 (m, 5H), 7.73-7.06 (m, 22H),6.53-6.25 (m, 3H), 6.13 (dq, J=51.3, 4.8 Hz, 1H), 5.95-5.46 (m, 3H),4.75-4.34 (m, 2H), 4.22-3.95 (m, 3H), 3.55-3.10 (m, 3H), 2.92 (t, J=5.9Hz, 1H), 2.55 (d, J=13.5 Hz, 1H), 2.32 (dt, J=11.3, 6.6 Hz, 1H); ¹⁹F NMR(282 MHz, DMSO-d₆) δ −204.01, −204.03, −204.33, −204.40; ³¹P NMR (121MHz, DMSO-d₆) δ 74.35, 74.01; LC/MS (ESI, m/z): [(M+1)]⁺=1118.0.

Step 4

O-((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl)O-(2-cyanoethyl)(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)-4-fluorotetrahydrofuran-2-yl)methyl)phosphoramidothioate(180)

To a solution ofO-((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl)O-(2-cyanoethyl)(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)phosphoramidothioate(179, 0.70 g, 0.63 mmol) in acetonitrile (5 mL) were added pyridiniumtrifluoroacetate (0.18 g, 0.94 mmol) and3-([bis[bis(propan-2-yl)amino]phosphanyl]oxy)propanenitrile (0.38 g,1.25 mol). The resulting solution was stirred for 2 hours at ambienttemperature. Upon completion, the resulting solution was applied onto areversed phase C18 column, eluted with 70%˜95% (25 min) acetonitrile inwater to afford the title compound 180 as a colorless solid (0.7 g,85%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.27 (s, 2H), 8.80-8.61 (m, 3H),8.11-8.04 (m, 5H), 7.70-7.07 (m, 22H), 6.57-6.26 (m, 3H), 6.26-5.98 (m,1H), 5.95-5.58 (m, 2H), 4.97 (dtt, J=22.5, 10.9, 6.8 Hz, 1H), 4.42 (dt,J=13.3, 4.0 Hz, 1H), 4.33-3.96 (m, 3H), 3.83 (dtd, J=14.6, 6.1, 3.8 Hz,2H), 3.76-3.57 (m, 2H), 3.32-3.13 (m, 2H), 2.96-2.67 (m, 4H), 2.54 (d,J=9.3 Hz, 1H), 2.40-2.23 (m, 1H), 1.22-1.17 (m, 12H); ¹⁹F NMR (282 MHz,DMSO-d₆) δ −200.71, −200.74, −201.16, −201.21, −201.64, −201.67,−201.77, −201.81, −201.99, −202.01, −204.16, −204.33, −204.44, −204.61;³¹P NMR (121 MHz, DMSO-d₆) δ 149.99, 149.93, 149.88, 149.85, 149.75,149.64, 149.54, 74.72, 74.52, 74.14, 74.11; LC/MS (ESI, m/z):[(M+1)]⁺=1318.0.

Step 5

O-(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-{[({[(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-{[(triphenylmethyl)amino]methyl}oxolan-3-yl]oxy}(2-cyanoethoxy)sulfanvlidene-λ⁵-phosphanyl)amino]methyl}-4-fluorooxolan-3-ylO-2-cyanoethyl phosphonothioate (181)

To a mixture ofO-((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl)O-(2-cyanoethyl)(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-3-(((2-cyanoethoxy)(diisopropylamino)phosphino)oxy)-4-fluorotetrahydrofuran-2-yl)methyl)phosphoramidothioate(180, 0.70 g, 0.53 mmol) and 1H-1,2,3,4-tetrazole (37.2 mg, 0.53 mmol)in acetonitrile (5 mL) was bubbled hydrogen sulfide for 1 min. Theresulting solution was sealed and stirred for 2 hours at ambienttemperature. Upon completion, the resulting solution was applied onto areversed phase C18 column, eluting with 70%˜95% (25 min) acetonitrile inwater to afford the title compound 181 as a colorless solid (580 mg,87%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.24 (s, 2H), 8.87-8.55 (m, 3H),8.20-7.90 (m, 5H), 7.75-6.98 (m, 21H), 6.57-5.44 (m, 8H), 4.72-4.32 (m,2H), 4.28-3.87 (m, 3H), 3.42 (s, 1H), 3.25 (d, J=27.1 Hz, 3H), 2.85 (dt,J=51.9, 5.8 Hz, 2H), 2.31 (s, 1H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ −202.87,−203.12, −203.34, −203.71, −203.95, −204.02, −204.35; ³¹P NMR (121 MHz,DMSO-d₆) δ 74.43, 74.07, 4.62, 4.62, −1.59, −1.67, −2.54, −2.71, −2.87;LC/MS (ESI, m/z): [(M+1)]⁺=1251.0.

Step 6

O-(2R,3R,4R,5R)-2-{[({[(2R,3R,4R,5R)-2-(aminomethyl)-5-(6-benzamido-9H-purin-9-yl)-4-fluorooxolan-3-yl]oxy}(2-cyanoethoxy)sulfanylidene-λ?-phosphanyl)amino]methyl}-5-(6-benzamido-9H-purin-9-yl)-4-fluorooxolan-3-ylO-2-cyanoethyl phosphonothioate (182)

A solution of0-(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-{[({[(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-{[(triphenylmethyl)amino]methyl}oxolan-3-yl]oxy}(2-cyanoethoxy)sulfanylidene-λ⁵-phosphanyl)amino]methyl}-4-fluorooxolan-3-ylO-2-cyanoethyl phosphonothioate (181, 0.58 g, 0.46 mmol) indichloromethane (10 mL) was treated with dichloroacetic acid (0.24 g,1.05 mmol) for 30 min at ambient temperature. The resulting solution wasused in the next step without any workup: LC/MS (ESI, m/z):[(M+1)]⁺=1009.2.

Step 7

N-{9-[(1R,6R,8R,9R,10R,15R,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-3,12-bis(2-cyanoethoxy)-9,18-difluoro-3,12-disulfanylidene-2,7,11,16-tetraoxa-4,13-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(183)

To the above reaction solution was added acetonitrile (100 mL) followedby triethylamine (20 mL) and carbon tetrachloride (20 mL). The resultingsolution was stirred for 1 h at ambient temperature. Upon completion,the resulting solution was concentrated under reduced pressure to affordthe crude title compound 183 which was used directly in the next stepwithout further purification: LC/MS (ESI, m/z): [(M+1)]⁺=1007.2.

Step 8

Diammonium[(1R,6R,8R,9R,10R,15R,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-difluoro-3,12-dioxo-12-sulfanidyl-2,7,11,16-tetraoxa-4,13-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-3-yl]sulfanide (77)

The above residue was treated with 30% solution of methanamine inethanol (5 mL) for 20 min at ambient temperature. The volatile organiccompounds were distilled out under reduced pressure. The residue waspurified by Prep-HPLC with the following conditions: Column: AtlantisPrep T3 OBD Column, 19×250 mm 10 um; Mobile Phase A: water (plus 10mmol/L of NH₄HCO₃), Mobile Phase B: ACN; Flow rate: 20 mL/min; Gradient:2% B to 6% B in 10 min; Detector: 254/210 nm; to afford the firstcompound 77 diastereomer as a colorless solid (10.6 mg, retention time:7.42 min): ¹H NMR (300 MHz, D₂O) δ 8.15 (s, 2H), 8.06 (s, 2H), 6.33 (d,J=22.2 Hz, 2H), 5.80 (dd, J=51.2, 4.8 Hz, 2H), 5.65-5.49 (m, 2H), 4.32(d, J=9.6 Hz, 2H), 3.49-3.32 (m, 2H), 3.13-2.99 (m, 2H); ¹⁹F NMR (282MHz, D₂O) δ, −195.73; ³¹P NMR (121 MHz, D₂O) δ 57.00; LC/MS (ESI, m/z):[(M−2NH₃−1)]⁻=690.9. And the slower peak at 8.25 min was a mixture oftwo isomers of 77 as a colorless solid (12.1 mg): ¹H NMR (300 MHz, D₂O)δ 8.37 (s, 0.34H), 8.14 (s, 1.7H), 7.92 (s, 1H), 7.86 (s, 1H), 6.26-5.91(m, 3H), 5.71-5.19 (m, 3H), 5.47-5.19 (m, 2H), 4.37 (dd, J=27.2, 9.7 Hz,3H), 3.54-3.38 (m, 2H), 3.14-2.92 (m, 2H); ¹⁹F NMR (282 MHz, D₂O) δ−195.78, −197.35; ³¹P NMR (121 MHz, D₂O) δ 59.32, 57.59; LC/MS (ESI,m/z): [(M−2NH₃−1)]⁻=690.9.

Diammonium(1R,6S,8R,9R,10R,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-difluoro-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate)

Step 1

(2R,3R,4R,5S)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl(2-cyanoethyl) phosphonate (183)

To a solution ofN-(9-((2S,3R,4R,5R)-3-fluoro-4-hydroxy-5-((tritylamino)methyl)tetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(INT-E, 1.00 g, 1.63 mmol) in acetonitrile (5 mL) were added pyridiniumtrifluoroacetate (0.47 g, 2.45 mmol) and3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (0.98 g, 3.26mmol) at ambient temperature. After stirring for 2 hours, another batchof pyridinium trifluoroacetate (0.63 g, 3.26 mmol) was added followed bythe addition of water (0.29 mL, 16.3 mmol). The resulting solution wasstirred for 1 h at ambient temperature. Upon completion, the resultingsolution was applied onto a reversed phase C18 column, eluted with5%˜80% (30 min) acetonitrile in water to afford the title compound 183as a colorless solid (0.58 g, 26%): ¹H NMR (400 MHz, DMSO-d₆) δ11.26-11.23 (m, 1H), 8.75-8.57 (m, 1H), 8.41-8.31 (m, 1H), 8.16-8.00 (m,2H), 7.70-7.56 (m, 3H), 7.43-7.39 (m, 6H), 7.33-7.23 (m, 6H), 7.19-7.16(m, 3H), 6.55-6.32 (m, 1H), 6.24-5.64 (m, 2H), 4.43-4.41 (m, 1H),4.32-4.24 (m, 2H), 3.25-2.91 (m, 2H), 2.53-2.51 (m, 1H), 2.42-2.27 (m,1H); ¹⁹F NMR (376 MHz, DMSO-d₆) δ −203.18; ³¹P NMR (162 MHz, DMSO-d₆) δ8.89; LC/MS (ESI, m z): [(M+1)]⁺=815.0.

Step 2

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl(2-cyanoethyl)(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)phosphoramidate(184)

To a mixture of(2R,3R,4R,5S)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl(2-cyanoethyl) phosphonate (100 mg, 0.14 mmol) andN-(9-((2S,3R,4R,5R)-5-(aminomethyl)-3-fluoro-4-hydroxytetrahydrofuran-2-yl)-9H-purin-6-yl)benzamide(183, 51 mg, 0.14 mmol) in N-methyl pyrrolidone (1 mL) were addedN,N-diisopropylethylamine (45 mg, 0.35 mmol) and carbon tetrachloride(53 mg, 0.34 mmol). The resulting solution was stirred for 4 hours atambient temperature. Upon completion, the resulting solution was appliedonto a silica gel column, eluted with 1%˜30% methanol in dichloromethaneto afford the title compound 184 as a colorless solid (113 mg, 75%): ¹HNMR (300 MHz, DMSO-d₆) δ 11.21 (s, 2H), 8.78-8.57 (m, 3H), 8.24-8.20 (m,1H), 8.12-7.96 (m, 4H), 7.71-7.46 (m, 7H), 7.47-7.28 (m, 6H), 7.33-7.03(m, 9H), 6.48-6.25 (m, 2H), 6.18-5.45 (m, 4H), 4.60-4.45 (m, 1H),4.38-4.30 (m, 1H), 4.15-3.96 (m, 4H), 3.40-3.00 (m, 2H), 2.91-2.69 (m,2H); ³¹P NMR (162 MHz, DMSO-d₆) δ 9.98, 9.93, 6.78; LC/MS (ESI, m/z):[(M+1)]⁺=1102.0.

Step 3

(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-{[({[(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-{[(triphenylmethyl)amino]methyl}oxolan-3-yl]oxy}(2-cyanoethoxy)phosphoryl)amino]methyl}-4-fluorooxolan-3-yl2-cyanoethyl phosphonate (185)

To a solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-((tritylamino)methyl)tetrahydrofuran-3-yl(2-cyanoethyl)(((2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-3-hydroxytetrahydrofuran-2-yl)methyl)phosphoramidate(184, 200 mg, 0.18 mmol) in acetonitrile (1 mL) were added3-([bis[bis(propan-2-yl)amino]phosphanyl]oxy)propanenitrile (109 mg,0.36 mmol) and pyridinium trifluoroacetate (53 mg, 0.27 mmol) at ambienttemperature. The resulting solution was stirred for another 2 hoursfollowed by the addition of the second batch of pyridiniumtrifluoroacetate (106 mg, 0.55 mmol) and water (32.7 mg, 1.8 mmol,). Theresulting solution was stirred for 30 min at ambient temperature andapplied onto a reversed phase C18 column, eluting with 0˜65% (30 min)acetonitrile in water to afford the title compound 185 as a colorlesssolid (153 mg, 69%): ¹H NMR (300 MHz, DMSO-d₆) δ 11.28-11.23 (m, 2H),8.82-8.61 (m, 3H), 8.31-8.19 (m, 1H), 8.12-7.99 (m, 4H), 7.72-7.06 (m,22H), 6.63-6.23 (m, 2H), 6.25-5.31 (m, 4H), 4.46-3.90 (m, 6H), 3.50-3.05(m, 4H), 3.05-2.70 (m, 4H); ¹⁹F NMR (282 MHz, DMSO-d₆) δ −202.92,−202.93, −202.96, −202.97, −203.12, −203.13, −203.68, −203.70, −203.73,−203.93, −203.96, −204.03; ³¹P NMR (121 MHz, DMSO-d₆) δ 9.98, 9.92,9.89, 9.84, 9.78, 9.04, 8.91, 8.84; LC/MS (ESI, m/z): [(M+1)]⁺=1219.4.

Step 4

(2R,3R,4R,5R)-2-{[({[(2R,3R,4R,5R)-2-(aminomethyl)-5-(6-benzamido-9H-purin-9-yl)-4-fluorooxolan-3-yl]oxy}(2-cyanoethoxy)phosphoryl)amino]methyl}-5-(6-benzamido-9H-purin-9-yl)-4-fluorooxolan-3-yl2-cyanoethyl phosphonate (186)

A solution of(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-2-{[({[(2R,3R,4R,5R)-5-(6-benzamido-9H-purin-9-yl)-4-fluoro-2-{[(triphenylmethyl)amino]methyl}oxolan-3-yl]oxy}(2-cyanoethoxy)phosphoryl)amino]methyl}-4-fluorooxolan-3-yl2-cyanoethyl phosphonate (185, 260 mg, 0.21 mmol) in dichloromethane (10mL) was treated with 2,2-dichloroacetic acid (109.3 mg, 0.85 mmol) for 2hours at ambient temperature. Upon completion, the resulting solutionwas concentrated under reduced pressure and the residue was used in thenext step without further purification: LC/MS (ESI, m/z):[(M+1)]⁺=977.1.

Step 5

N-{9-[(1R,6S,8R,9R,10R,15S,17R,18R)-17-(6-benzamido-9H-purin-9-yl)-3,12-bis(2-cyanoethoxy)-9,18-difluoro-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecan-8-yl]-9H-purin-6-yl}benzamide(187)

To a solution of the compound 186 in acetonitrile (40 mL) were addedtriethylamine (0.75 mL, 5.34 mmol) and carbon tetrachloride (0.82 g,5.34 mmol) at ambient temperature. After stirring for 2 hours, theresulting solution was concentrated under reduced pressure and theresidue was used in the next step without further purification: LC/MS(ESI, m/z): [(M+1)]⁺=975.5.

Step 6

Diammonium(1R,6S,8R,9R,10R,15S,17R,18R)-8,17-bis(6-amino-9H-purin-9-yl)-9,18-difluoro-3,12-dioxo-4,7,13,16-tetraoxa-2,11-diaza-3λ⁵,12λ⁵-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-bis(olate)(78)

The above crude compound 187 was treated with a solution of methylaminein ethanol (10 mL, 30%, w/w) for 30 min at ambient temperature. Uponcompletion, the resulting solution was concentrated under reducedpressure and the residue was purified by Prep-HPLC with the followingconditions: Column: Atlantis Prep T3 OBD Column, 19×250 mm, 10 um;Mobile Phase A: water (plus 20 mmol/L of NH₄HCO₃), Mobile Phase B: ACN;Flow rate: 20 mL/min; Gradient: 0% B to 14% B in 20 min; Detector:254/220 nm; Retention time: 11.50 min; to afford the title compound 78as a colorless solid (3.7 mg, 3%): ¹H NMR (300 MHz, D₂O) δ 8.12 (s, 2H),7.65 (s, 2H), 6.23 (d, J=20.7 Hz, 2H), 5.52-5.22 (m, 4H), 4.36 (d, J=9.3Hz, 2H), 3.47 (dd, J=13.9, 3.3 Hz, 2H), 3.08 (dd, J=13.8, 7.3 Hz, 2H);¹⁹F NMR (282 MHz, D₂O) 6-196.79; ³¹P NMR (121 MHz, D₂O) δ 7.98; LC/MS(ESI, m/z): [(M−2NH₃−1)]⁻=659.0

Biological Assays

STING pathway activation by the compounds described herein was measuredusing THP1-Dualtm cells. These cells are THP1 monocytes that have beenmodified to be reporters for the NFκB pathway (by inducing secretedembryonic alkaline phosphatase (SEAP) expression) and the IRF pathway(by inducing secreted luciferase (LUCIA)). Both of these pathways areactivated by STING agonists in these cells.

THP1 Dualtm cells (obtained from Invivogen) are maintained in a cellgrowth medium that includes Roswell Park Memorial Institute medium(RPMI), 10% fetal calf serum (FCS), 100 U/ml Pen/Strep, 2 mM L-glut, 10mM Hepes, and 1 mM sodium pyruvate. Prior to the assay, the cells weretransferred to an assay medium that includes RPMI, 5% FCS, 100 U/mlPen/Strep, 2 mM L-glut, 10 mM Hepes, and 1 mM sodium pyruvate. Cellswere then counted and evaluated for viability by trypan blue exclusionassay.

Compounds were dissolved in water or DMSO depending, for example, ontheir solubility in water or DMSO. The compounds are then diluted in theassay medium and plated into wells of a 384-well tissue culture plate in25 μL portions. Cells are then added in 25 μL assay medum to result in afinal cell concentration of 80,000 cells per well.

For each set of compounds, two plates were prepared: one plate that wassubjected to a 24-hour assay duration, and one plate that was subjectedto a 48-hour assay duration. The plates are incubated during theirrespective assay durations at 37° C., with 5% CO₂.

To carry out the secreted embryonic alkaline phosphatase reporter, 10 μLof cell supernatant was mixed with 90 μL of QUANTI-Blue in a flat-bottom384 well plate. The plates were incubated at 37° C. for 1-2 hours. SEAPactivity was measured using a spectrophotometer set at 620 nm. In thesecreted luciferase (i.e., Lucia) assay, 10 μL of THP1-Blue™ WASG cellsupernatant was plated, then 50 μL Quanti LUC Solution was added.Luminescence of the wells was then measured.

Table 2 below depicts biological data of the compounds that were assayedusing the above procedures.

TABLE 2 IRF3 NFκB (EC₅₀ Compound (EC₅₀ μM) μM) 71 1-10 1-10 72-0A1 1-10<1 72-0A2 1-10 1-10 72-0A3 1-10 <1 72-0B1 1-10 50-100 72-0B2 10-49 10-49  73 10-49  1-10 74-0A1 1-10 1-10 74-0A2 1-10 <1 74-0B0 10-49 10-49  74-0B1 10-49  50-100 75 10-49  10-49  76-0A1 10-49  1-10 76-0A210-49  10-49  76-0A3 1-10 <1 76-0B1 10-49  50-100 77 50-100 50-100 7850-100 50-100

Compounds can also be assayed using the procedures described in, e.g.,WO 2015/077354.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

1-276. (canceled)
 277. A compound of the formula

wherein X¹ and X⁵ are each independently halo or —OH; Y¹ and Y² are eachindependently —OH or —SH; and A and B are each independently

or a stereoisomer or a pharmaceutically acceptable salt thereof. 278.The compound according to claim 277 which is

or a stereoisomer or a pharmaceutically acceptable salt thereof. 279.The compound according to claim 278 which is

or a stereoisomer or a pharmaceutically acceptable salt thereof. 280.The compound according to claim 278 which is

or a stereoisomer or a pharmaceutically acceptable salt thereof.
 281. Acompound which is


282. The compound according to claim 278 which is

or a stereoisomer or a pharmaceutically acceptable salt thereof.
 283. Acompound which is


284. A compound which is

or a stereoisomer or a pharmaceutically acceptable salt thereof.
 285. Acompound which is


286. A compound which is

or a stereoisomer or a pharmaceutically acceptable salt thereof.
 287. Acompound which is


288. A compound which is


289. A compound which is


290. A compound which is


291. A compound which is


292. A compound selected from(1S,3R,6S,8R,9R,10S,12R,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-disulfanyl-4,7,13,16-tetraoxa-2,11-diaza-3lambda5,12lambda5-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-dione;(1S,3S,6S,8R,9R,10S,12R,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-disulfanyl-4,7,13,16-tetraoxa-2,11-diaza-3lambda5,12lambda5-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-dione;(1S,3S,6S,8R,9R,10S,12S,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-disulfanyl-4,7,13,16-tetraoxa-2,11-diaza-3lambda5,12lambda5-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-dione;(1S,3R,6S,8R,9R,10S,12S,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-9,18-dihydroxy-3,12-disulfanyl-4,7,13,16-tetraoxa-2,11-diaza-3lambda5,12lambda5-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-dione;or(1S,6S,8R,9R,10S,15S,17R,18R)-8-(2-amino-6-oxo-6,9-dihydro-1H-purin-9-yl)-17-(6-amino-9H-purin-9-yl)-3,9,12,18-tetrahydroxy-4,7,13,16-tetraoxa-2,11-diaza-3lambda5,12lambda5-diphosphatricyclo[13.3.0.0^(6,10)]octadecane-3,12-dione.293. A pharmaceutical composition comprising a compound according toclaim 277 or a pharmaceutically acceptable salt thereof and one or morepharmaceutically acceptable carriers, diluents or excipients.
 294. Acombination pharmaceutical product comprising a compound according toclaim 277 or a pharmaceutically acceptable salt thereof together withone or more other therapeutically active agents.
 295. A compoundaccording to claim 277 or a pharmaceutically acceptable salt thereof foruse in therapy.
 296. A compound according to claim 277 or apharmaceutically acceptable salt thereof for use in the treatment ofdiseases or conditions that may be alleviated by the induction of animmune response via the STING pathway.
 297. A compound or apharmaceutically acceptable salt thereof for use according to claim 277,wherein the disease or condition is cancer.
 298. The use of a compoundaccording to claim 277 or a pharmaceutically acceptable salt thereof, inthe manufacture of a medicament for the treatment of diseases andconditions in which the modulation of STING is indicated.
 299. A methodof treating diseases and conditions in which the modulation of STING isindicated in a subject in need thereof which comprises administering atherapeutically effective amount of compound according to claim 277 or apharmaceutically acceptable salt thereof.
 300. A method of treatingcancer comprising administering a therapeutically effective amount ofone or more compounds according to claim 277 or a pharmaceuticallyacceptable salt thereof.
 301. The method of claim 300 wherein the canceris small cell lung cancer, non-small cell lung cancer, colorectalcancer, melanoma, renal cell carcinoma, head and neck cancer, Hodgkin'slymphoma, bladder cancer, esophageal carcinoma, gastric carcinoma,ovarian carcinoma, cervical carcinoma, pancreatic carcinoma, prostatecarcinoma, breast cancers, urinary carcinoma, brain tumors such asglioblastoma, non-Hodgkin's lymphoma, acute lymphatic leukemia (ALL),chronic lymphatic leukemia (CLL), acute myeloid leukemia (AML), chronicmyeloid leukemia (CML), hepatocellular carcinoma, multiple myeloma,gastrointestinal stromal tumors, mesothelioma, and other solid tumors orother hematological cancers.
 302. The method of claim 301 wherein thecancer is small cell lung cancer, non-small cell lung cancer, colorectalcancer, melanoma, renal cell carcinoma, head and neck cancer, Hodgkin'slymphoma or bladder cancer.
 303. A method for treating cancer in asubject in need thereof, comprising administering an effective amount ofa compound, according to claim 277, or a pharmaceutically acceptablesalt thereof, in combination with the administration of atherapeutically effective amount of one or more immuno-oncology agents.304. The method of claim 303, wherein the immuno-oncology agent isipilimumab.
 305. The method of claim 303, wherein the immuno-oncologyagent is a PD-L1 antagonist.
 306. A method for treating a subjectafflicted with cancer comprising administering to the subject atherapeutically effective amount of: a) a compound according to claim277, or a pharmaceutically acceptable salt thereof, and b) ananti-cancer agent which is an antibody or an antigen-binding portionthereof that binds specifically to a Programmed Death-1 (PD-1) receptorand inhibits PD-1 activity.
 307. The method of claim 306, wherein theanti-PD-1 antibody is nivolumab or pembrolizumab.
 308. The method ofclaim 307, wherein the anti-PD-1 antibody is nivolumab.